Immunomodulatory polynucleotides in treatment of an infection by an intracellular pathogen

ABSTRACT

The present invention features methods for treatment or prevention of infection by intracellular pathogens (e.g., Mycobacterium species) by administration of an immunomodulatory nucleic acid molecule. In one embodiment, immunomodulatory nucleic acid molecule are administered in combination with another anti-pathogenic agent to provide a synergistic anti-pathogenic effect.

CROSS-REFERENCE To RELATED APPLICATIONS

[0001] This application is claims the benefit of U.S. provisional patentapplication No. 60/179,353, filed Jan. 31, 2000, which is incorporatedherein by reference in its entirety.

STATEMENT As To FEDERALLY SPONSORED RESEARCH

[0002] This invention was made, at least in part, with a governmentgrant from the National Institutes of Health (NIH Grant Nos. A140682,A147078, and HL57911). Thus, the U.S. government may have certain rightsin this invention.

FIELD OF THE INVENTION

[0003] The invention relates generally to the field of prevention andtreatment of infectious diseases, particularly infection byintracellular pathogens such as Mycobacterium.

BACKGROUND OF THE INVENTION

[0004] The broad classification of intracellular pathogens includesviruses, bacteria, protozoa, fungi, and intracellular parasites. Thesevirulent pathogens multiply within the cells of the infected hostorganism rather than extracellularly and are major causes of morbidityand fatality world-wide. For example, intracellular pathogens areresponsible for an estimated 10,000,000 new cases of tuberculosis peryear in the world (approximately 25,000 per year in the United States),approximately 3,000,000 deaths per year from tuberculosis, an estimated12,000,000 cases of leprosy, and an estimated 10,000,000 cases ofAmerican trypanosomiasis (Chagas disease). Furthermore, intracellularpathogens also cause other important diseases including cutaneous andvisceral leishmaniasis, listeriosis, toxoplasmosis, histoplasmosis,trachoma, psittacosis, Q-fever, and Legionellosis includingLegionnaires' disease. Few vaccines are available against such diseasesand the pathogens are developing resistance to commonly used drugs.

[0005] One particular genus of intracellular bacteria, Mycobacteria, isa significant cause of morbidity and mortality, particularly amongimmunocompromised or elderly individuals and in countries with limitedmedical resources. Ninety-five percent of human infections are caused byseven species: Mycobacterium tuberculosis, M. avium (also known as themycobacterium avium complex or M. avium-intracellulare), M. leprae, M.kansasii, M. fortuitum, M. chelonae, and M. absecessus. The most commonmycobacterial infections in the United States are pulmonary infectionsby M. tuberculosis or M. avium. Such mycobacterial infections have beenof increasing concern over the past decade, particularly in light of theincreasing incidence of multi-drug resistant strains.

[0006]M. tuberculosis is the causative agent of tuberculosis, theclassic human mycobacterial disease. Disease is spread by closeperson-to-person contact through inhalation of infectious aerosols;infection can be established if as few as one to three bacilli reach thealveolar spaces. Estimates indicates that one-third of the world'spopulation, including 10 million in the U.S., are infected with M.tuberculosis, with 8 million new cases and 3 million deaths reportedworld wide each year. Although incidence of tuberculosis steadilydecreased since the early 1900s, this trend changed in 1984 withincreased immigration from endemic countries and increased infection inthe homeless, drug and alcohol abusers, prisoners, and HIV-infectedindividuals ((1995) Morbid. Mortal. Weekly Rep 44:1-87). Due to thedifficulties in eradicating disease in most of these populations,tuberculosis has again threatened to pose a significant public healthrisk.

[0007]Mycobacterium avium is generally less of a health risk forindividuals with normal immune responses; M. avium can transientlycolonize these individuals, but disease due to M. avium is rare.However, M. avium infection can cause serious disease in patients havingcompromised pulmonary function (e.g., patients with chronic bronchitisobstructive pulmonary disease, or pre-existing pulmonary damage (e.g.,due to previous pulmonary infections or other disease). Infection inindividuals having compromised pulmonary function is clinically verysimilar to infection by M. tuberculosis.

[0008]M. avium infection poses the greatest health risk toimmunocompromised individuals, and is one of the most commonopportunistic infections in patients with AIDS (Horsburgh (1991) NewEng. J. Med. 324:1332-1338). In contrast with disease in other patients,M. avium infection can be very serious in immunocompromised individuals(e.g., AIDS patients, who have a low CD4+ T-cell count (Crowe, et al.(1991) J. AIDS 4:770-776)), and can result in disseminated infection inwhich virtually no organ is spared. The magnitude of such disseminatedM. avium infections is overwhelming, with the bacterial load in somepatients resulting in tissues that are literally filled withmycobacteria and with hundreds to thousands of bacilli per milliliter ofblood. When disseminated disease occurs, M. avium infection results inconsiderable morbidity, and is a significant contributor to mortality inAIDS patients. Although highly active anti-retroviral therapy currentlyused to treat HIV-infected patients prevents the onset of M. aviuminfection to some extent (Autran, et al. (1997) Science. 277:112-116),this infection is extremely difficult to treat when encountered becauseof its poor responsiveness to anti-mycobacterial therapy (Chin, et al.(1994) J. Infect. Dis. 170:578-584; Masur (1993) New Eng. J. Med.329:898-904).

[0009] As noted above, mycobacterial infection is normally acquiredthrough inhalation of aerosolized infectious particles. Followinginhalation, mycobacteria predominately infect and multiply withinmacrophages (Edwards, et al. (1986) Am. Rev. Respir. Dis.134:1062-1071). The bacteria attach to and enter macrophages with thehelp of specific receptors expressed on the surface of these cells(Bermudez, et al. (1991) Infect. Immun. 59:1697-1702; Rao, et al. (1993)Infect. Immun. 61:663-670; Roecklein, et al. (1992) J. Lab. Clin. Med.119:772-781). Studies have shown that macrophages secrete severalcytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β,IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), andgranulocyte colony stimulating factor (Fattorini, et al. (1994) J. Med.Microbiol. 40:129-133; Newman, et al. (1991) J. Immunol. 147:3942-3948)in response to infection with mycobacteria. T cell products such asinterferon (IFN)-y and IL-12 are known to be extremely important foranti-mycobacterial activity of macrophages (Fattorini, et al. (1994) J.Med. Microbiol. 40: 129-133) as well as in vivo in humans and mice(Appelberg, et al. (1994) Infect. Immun. 62:3962-3971; Holland, et al.(1994) New Eng. J. Med. 330:1348-1355; Kobayashi, et al. (1995)Antimicrob. Agents Chemotherapy. 39:1369-1371).

[0010] Treatment of mycobacterial infections is complicated anddifficult. For example, treatment of M. tuberculosis and of M. aviuminfections requires a combination of relatively toxic agents, usuallythree different drugs, for at least six months. The toxicity andintolerability of these medications usually result in low compliance andinadequate treatment, which in turn increases the chance of therapeuticfailure and enhances the selection for drug-resistant organisms.Treatment of mycobacterial infections is further complicated in pregnantwomen, patients with pre-existing liver or renal diseases, andimmunocompromised patients, e.g., AIDS patients.

[0011] Immunomodulatory sequences (hereinafter referred to as “ISS”)were initially discovered in the mycobacterial genome as DNA sequencesthat selectively enhance NK cell activity (Yamamoto, et al. (1992)Microbiol. Immunol. 36:983-997). Uptake of mycobacterial DNA or ISS hasbeen shown to activate cells of the innate immune system, such as NKcells and macrophages and stimulating a type-i like response (Roman, etal. (1997) Nature Med. 3:849-854). Further, administration of ISS hasbeen shown activate NK cells (Krieg, A et al. (1995) Nature.374:546-549), stimulate B cells to proliferate and to produce IgMantibodies (Krieg, A et al. (1995) Nature. 374:546-549; Messina, et al.(1991) J. Immunol. 147:1759-1764;), stimulate production of cytokines,such as IFNs, IL-12; IL-18 and TNF-α (Sparwasser, et al. (1998) Eur. J.Immunol. 28:2045-2054; Sparwasser, et al. (1997) Eur. J. Immunol.27:1671-1679; Stacey, et al. (1999) Infect. Immun. 67:3719-3726; Stacey,et al. (1996) J. Immunol. 157:2116-2122; Halpern, et al (1996) Cell.Immunol. 167:72-78; Klinman, et al. (1996) Proc. Natl. Acad. Sci. U.S.A.93:2879-2883) and up-regulate co-stimulatory receptors (Martin-Orozco,et al. (1999) Int. Immun. 11:1111-1118; Sparwasser, et al. (1998) Eur.J. Immol. 28:2045-2054).

[0012] Previous studies have demonstrated the ability ofimmunomodulatory nucleic acid to enhance innate immunity and hostsurvival against intracellular pathogens such as Listeria monocytogenes,Leishmania major, and Francisella tularensis (Krieg, et al. (1998) J.Immunol. 161:2428-2434; Walker, et al. (1999) Proc. Natl. Acad. Sci.U.S.A. 96:6970-6975; Zimmermann, et al. (1998) J. Immunol.160:3627-3630; Klinman, et al. (1999) Infect. Immun. 67:5658-5663).Walker, et al. found that injection of BALB/c mice with CpG-ODN 1826four hours after inoculation with live L. major promastigote organismsprotected 65% of animals tested from progressive infection, suggestingthat CpG-ODN can redirect the harmful immune response elicited by liveL. major parasites and that CpG-ODN might be efficacious in thetreatment of early leishmaniasis (Walker, et al. (1999) Proc. Natl.Acad. Sci. U.S.A. 96:6970-6975). Zimmermann et al. report that singleinjections of CpG-ODN protected L. major-infected BALB/c mice when givenduring the first 8 days of infection but failed when given later.Zimmermann also found that 5 of 6 L. major-infected BALB/c mice wereable to control the infection when given three consecutive doses ofCpG-ODN at 5 day intervals starting on day 15 or 20 post infection(Zimmermann, et al. (1998) J. Immunol. 160:3627-3630).

[0013] In their studies with L. monocytogenes, Kreig, et al. found thatIFN-γ production is induced rapidly by ISS administration, returning tothe basal level within 24 hours, while IL-12 (p40 and p70) is inducedimmediately after infection and lasts for at least 8 days (Krieg, et al.(1998) J. Immunol. 161:2428-2434). In the murine leishmaniasis model,the serum IL-12 level in the ISS-treated mice was found to be 10-foldhigher than L. major-infected control mice (Zimmermann, et al. (1998) J.Immunol. 160:3627-3360).

[0014] Exogenous administration of type-1 cytokines, such as IL-12 andIFN-γ increase protection against M. avium infection in humans and mice(Appelberg, et al. (1994) Infect. Immun. 62:3962-3971; Holland, et al.(1994) New Eng. J. Med. 330:1348-1355; Kobayashi, et al. (1995)Antimicrob. Agents Chemotherapy. 39:1369-1371). IFN-γ and IL-12 areknown to be important in host anti-mycobacterial immunity (Doherty, etal. (1997) J. Immunol. 158:4822-4831; Doherty,et al. (1998) J. Immunol.160:5428-5435). However, administration of such cytokines is potentiallydangerous to the patient, is expensive and does not provide anattractive means of preventing or treating existing infections byintracellular pathogens. Furthermore, administration of these cytokinescan itself be associated with undesirable side-effects which are due atleast in part to toxicity, especially at dosages sufficient to stimulatethe subject's immune system.

[0015] DNA vaccines may provide an alternative method for therapy. DNAvaccination with a plasmid which encodes M. avium antigens (65 kDa andantigen 85B) had a protective effect against M. avium infection in mice(Velaz-Faircloth, et al. (1999) Infect. Immun. 67:4243-4250). Similarly,plasmid DNA which encodes antigen 85B, ESAT-6 and MPT64 (Kamath, et al.(1999) Infect. Immun. 67:1702-1707), and hsp-65 (Bonato, et al. (1998)Infect. Immun. 66:169-175) yielded protective immunity against M.tuberculosis infection. Further DNA vaccines based upon administrationof a polynucleotide encoding a mycobacterial antigen are described in WO98/53075. However, while these methods appear promising, DNA vaccinationrequires identification of an antigen that will induce a protectiveimmune response. Furthermore, the immune response elicited by thesevaccines is predominantly a type-1 response (i.e., mediated by Th1 cellsand primarily resulting in production of antibodies). As discussedabove, a robust cellular immune type-I immune response (i.e., an immuneresponse mediated by Th1 cells and primarily resulting in activation ofcytotoxic T cells, which secrete IFN-γ) is likely required to provideeffective immunity against such intracellular pathogens. Finally, whileDNA vaccines may provide some protection against infection in apreventive mode, their effectiveness against an ongoing infection is notproven.

[0016] There remains a need in the field for effective methods for thetreatment and prevention of infection by intracellular pathogens.

SUMMARY OF THE INVENTION

[0017] The present invention features methods for treatment orprevention of infection by intracellular pathogen by administration ofan immunomodulatory nucleic acid molecule (ISS). In one embodiment, ISSare administered in combination with another anti-pathogenic agent toprovide a synergistic anti-pathogenic effect. In a preferred embodiment,the intracellular pathogen is Mycobacterium species.

[0018] A primary object of the invention is to provide an effectivemethod for the prevention and/or treatment of intracellular pathogeninfections in a host, particularly mycobacterial infections.

[0019] Another object of the invention is to enhance the anti-pathogenicactivity, particularly the anti-mycobacterial activity, of conventionalchemotherapeutics to facilitate more effective clearance of the organismfrom an active infection in a subject.

[0020] One advantage of the invention is that, since immunomodulatorynucleic acid molecules act through induction of the immune response ofthe host, the use of immunomodulatory nucleic acid molecules will notsubstantially result in the selection of resistant organisms. Stillanother advantage is that immunomodulatory nucleic acids acts in synergywith conventional antibiotics, particularly in the context of treatmentof mycobacterial infection.

[0021] These and other objects and advantages will be readily apparentto the ordinarily skilled artisan upon reading the disclosure providedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a graph showing the effect of immunomodulatory nucleicacid molecules (ISS), exemplified here by immunomodulatory DNAoligonucleotides (ISS-ODN), on intracellular growth of M. avium in humanmonocyte-derived macrophages (hMDM) in vitro. Results shown arerepresentative of three experiments. Closed circles, medium alone; smalldiamonds, mutated ISS; inverted triangle, ISS at 3 μg/ml; closed square,ISS at 10 μg/ml; and closed triangle, ISS at 30 μg/ml.

[0023]FIG. 2 is a graph showing the effect of ISS upon infected hMDM.ISS, ISS added; medium, medium alone.

[0024]FIG. 3 is a graph showing the effect of ISS and antibiotics uponinfected hMDM. ISS, ISS; CLA, clarithromycin.

[0025] FIGS. 4A-4B are graphs showing the effect of ISS, exemplifiedhere by ISS-ODN, on intracellular growth of M. avium in hMDM in vitro(FIG. 4A) and effect of ISS and antibiotics upon infected hMDM (FIG.4B). Closed square, medium alone; closed circle, mutated ISS; closedtriangle, ISS. ISS, ISS-ODN; M-ODN, mutated-ODN; CLA, clarithromycin.Results shown are mean±SD for three experiments. *p<0.01 compared to CFUrecovered from cells treated with M-ODN or medium alone.

[0026] FIGS. 5A-5C are graphs showing the effect of ISS and antibioticson mBMDM in vivo (FIG. 5A) and in vitro (FIGS. 5B-5D). Results shown aremean±SD for three experiments. *p<0.05 compared to CFU in the organs ofcontrol mice that received CLA only or a combination of CLA and M-ODN.

[0027]FIG. 6 is a graph showing the effect of ISS, exemplified here byISS-ODN, on M. avium (106 organisms/mouse) growth in C57B1/6 mice.Results shown are mean±SD of the number of CFU per organ (spleen, toppanel; lung, bottom panel). ISS,closed circles; PBS (control), trianglesand dashed lines. *p<0.05 compared to CFU in the organs of control micethat received PBS instead of ISS. Results shown are mean±SD.

[0028] FIGS. 7A-7C are graphs showing the effect of ISS, exemplifiedhere by ISS-ODN, on M. avium (107 organisms/mouse) growth in C57B1/6mice. Results shown are mean±SD of the number of CFU per organ (spleen,FIG. 7A; lung, FIG. 7B; liver, FIG. 7C). ISS, closed circles; PBS(control), triangles and dashed lines. *p<0.05 compared to CFU in theorgans of control mice that received PBS instead of ISS. Results shownare mean±SD.

[0029]FIG. 8 is a graph showing IFN-γ production in M. avium-infectedmice pre-treated with ISS, exemplified here by ISS-ODN. Levels at weeks2, 4, and 6 are shown in left, center, and right panels, respectively.Uninfected, closed circles; PBS-treated control, open circles; ISS-ODNtreated, closed triangles.

[0030] FIGS. 9A-9B are graphs showing the effect of ISS on intracellulargrowth of M. avium in mBMDM in vitro on days 1, 3, and 7 after infection(FIG. 9A) and 7 days after infection (FIG. 9B). Closed square, mediumalone; closed circle, mutated ISS; closed triangle, ISS. Results shownare mean±SD for triplicate experiments. *p<0.01 compared to CFUrecovered from cells treated with M-ODN or medium alone.

[0031] FIGS. 10A-10C are graphs showing the CD4⁺ (FIG. 10A), CD8⁺ (FIG.10B), and IFN-γ⁺ (FIG. 10C) T cell responses of mice treated with eitherISS or M-ODN prior to infection. Splenocytes were pooled within groupsfor intracellular IFN-γ assays (panels A, B). For total IFN-γ produced,results represent the mean±SD (panel C).

[0032]FIGS. 11A and 11B are schematics, and FIG. 11C a graph, showingthe effect of ISS, exemplified here by ISS-ODN, on indoleamine2,3-dioxygenase (IDO) in mouse cells. FIG. 11A represents results ofsemi-quantitative RT-PCR assessment of IDO induction in mice 16 hoursafter ISS injection. −, injected with saline; +, injected with ISS-ODN.FIG. 11B represents results of semi-quantitative RT-PCR assessment ofIDO induction in ISS-ODN pre-treated mBMDM 4, 8, and 24 hours afterinfection with M. avium. 1, medium alone; 2, ISS-ODN treatment alone; 3,M. avium infection alone; 4, ISS-ODN treatment and M. avium infection.FIG. 11C represents inhibition of ISS induction of IDO by L-tryptophanor 1-methyl-DL-tryptophan (a competitive IDO inhibitor). L-Try,L-tryptophan; M-Try, 1-methyl-DL-tryptophan. Results shown are mean±SDfor triplicate experiments. *p<0.01 compared to CFU recovered from cellstreated with ISS-ODN.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Before the present invention is described, it is to be understoodthat this invention is not limited to particular embodiments described,as such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

[0034] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

[0035] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of such cells and reference to “thepolynucleotide” includes reference to one or more polynucleotides andequivalents thereof known to those skilled in the art, and so forth.

[0036] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

[0037] Definitions

[0038] As used herein, the term “pathogen” or “intracellular pathogen”or “microbe” refers to any organism that exists within a host cell,either in the cytoplasm or within a vacuole, for at least part of itsreproductive or life cycle. Intracellular pathogens include viruses(e.g., CMV HIV), bacteria (e.g., Listeria, Mycobacteria, Salmonella(e.g., S. typhi) enteropathogenic Escherichia coli (EPEC),enterohaemorrhagic Escherichia coli (EHEC), Yersinia, Shigella,Chlamydia, Chlamydophila, Staphylococcus, Legionella), protozoa (e.g.,Taxoplasma), fungi, and intracellular parasites (e.g., Plasmodium (e.g.,P. vivax, P. falciparum, P. ovale, and P. malariae).

[0039] The terms “immunomodulatory nucleic acid molecule,” “ISS,”“ISS-PN,” and “ISS-ODN, “used interchangeably herein, refer to apolynucleotide that comprises at least one immunomodulatory nucleic acidmoiety. The term “immunomodulatory, “as used herein in reference to anucleic acid molecule, refers to the ability of a nucleic acid moleculeto modulate an immune response in a vertebrate host.

[0040] The terms “oligonucleotide,” “polynucleotide,” and “nucleic acidmolecule”, used interchangeably herein, refer to polymeric forms ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Thus, this term includes, but is not limited to,single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the polynucleotide cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.Alternatively, the backbone of the polynucleotide can comprise a polymerof synthetic subunits such as phosphoramidites, and/orphosphorothioates, and thus can be an oligodeoxynucleosidephosphoramidate or a mixed phosphoramidate-phosphodiester oligomer.Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al.(1996) Nucl. Acids Res. 24:2318-2323. The polynucleotide may compriseone or more L-nucleosides. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars, and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may bemodified to comprise N3′-P5′ (NP) phosphoramidate, morpholinophosphorociamidate (MF), lockaed nucleic acid (LNA), 2′-O-methoxyethyl(MOE), or 2′-fluoro, arabino-nucleic acid (FANA), which can enhance thereistance of the polynucleotide to nuclease degradation (see, e.g.,Faria et al. (2001) Nature Biotechnol. 19:40-44; Toulme (2001) NatureBiotechnol. 19:17-18). A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications included in this definition are caps,substitution of one or more of the naturally occurring nucleotides withan analog, and introduction of means for attaching the polynucleotide toproteins, metal ions, labeling components, other polynucleotides, or asolid support. Immunomodulatory nucleic acid molecules can be providedin various formulations, e.g., in association with liposomes,microencapsulated, etc., as described in more detail herein.

[0041] The terms “polypeptide,” “peptide,” and “protein”, usedinterchangeably herein, refer to a polymeric form of amino acids of anylength, which can include coded and non-coded amino acids, chemically orbiochemically modified or derivatized amino acids, and polypeptideshaving modified peptide backbones. The term includes polypeptide chainsmodified or derivatized in any manner, including, but not limited to,glycosylation, formylation, cyclization, acetylation, phosphorylation,and the like. The term includes naturally-occurring peptides, syntheticpeptides, and peptides comprising one or more amino acid analogs. Theterm includes fusion proteins, including, but not limited to, fusionproteins with a heterologous amino acid sequence, fusions withheterologous and homologous leader sequences, with or without N-terminalmethionine residues; immunologically tagged proteins; and the like.

[0042] As used herein the term “isolated” is meant to describe acompound of interest that is in an environment different from that inwhich the compound naturally occurs. “Isolated” is meant to includecompounds that are within samples that are substantially enriched forthe compound of interest and/or in which the compound of interest ispartially or substantially purified.

[0043] As used herein, the term “substantially purified” refers to acompound that is removed from its natural environment and is at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which it is naturally associated.

[0044] “Treatment” or “treating” as used herein means any therapeuticintervention in a subject, usually a mammalian subject, generally ahuman subject, including: (i) prevention, that is, causing the clinicalsymptoms not to develop, e.g., preventing infection and/or preventingprogression to a harmful state; (ii) inhibition, that is, arresting thedevelopment or further development of clinical symptoms, e.g.,mitigating or completely inhibiting an active (ongoing) infection sothat pathogen load is. decreased to the degree that it is no longerharmful, which decrease can include complete elimination of aninfectious dose of the pathogen from the subject; and/or (iii) relief,that is, causing the regression of clinical symptoms, e.g., causing arelief of fever, inflammation, and/or other symptoms caused by aninfection.

[0045] As used herein, “immunoprotective response” is meant to encompasshumoral and/or cellular immune responses that are sufficient to: 1)inhibit or prevent infection by an intracellular pathogen, particularlyMycobacteria; and/or 2) prevent onset of disease, reduce the risk ofonset of disease, or reduce the severity of disease symptoms caused byinfection by an intracellular pathogen, particularly Mycobacteria.

[0046] The term “effective amount” or “therapeutically effective amount”means a dosage sufficient to provide for treatment for the disease statebeing treated or to otherwise provide the desired effect (e.g.,induction of an effective immune response). The precise dosage will varyaccording to a variety of factors such as subject-dependent variables(e.g., age, immune system health, etc.), the disease (e.g., the speciesof the infecting pathogen), and the treatment being effected. In thecase of an intracellular pathogen infection, an “effective amount” isthat amount necessary to substantially improve the likelihood oftreating the infection, in particular that amount which improves thelikelihood of successfully preventing infection or eliminating infectionwhen it has occurred.

[0047] By “subject” or “individual” or “patient” is meant any subjectfor whom or which therapy is desired. Human subjects are of particularinterest. Other subjects may include non-human primates, cattle, sheep,goats, dogs, cats, birds (e.g., chickens or other poultry), guinea pigs,rabbits, rats, mice, horses, and so on. Of particular interest aresubjects having or susceptible to intracellular pathogen infection,particularly mycobacterial infection, more particularly to infection byM. tuberculosis, M. avium, and the like.

[0048] Overview

[0049] The invention is based on the discovery that: 1) administrationof immunomodulatory nucleic acid molecules results in induction of animmune response protective against infection by mycobacteria; 2)immunomodulatory nucleic acid molecules act as a chemotherapeutic agent(as evidenced by, for example, the ability of immunomodulatory nucleicacid molecules to inhibit growth of mycobacteria when administeredalone); 3) immunomodulatory nucleic acid molecules provide a synergisticeffect when administered with another chemotherapeutic agent; and 4)administration of immunomodulatory nucleic acid molecules results ininduction of indoleamine 2,3-dioxygenase (IDO), indicatingimmunomodulatory nucleic acid molecules have activity against a widerange of intracellular pathogens that utilize L-tryptophan of the hostcell. In short, immunomodulatory nucleic acid molecule administrationresults in activation of the subject's innate immunity and induction ofIDO synthesis, takes advantage of chemotherapeutic activity of theimmunomodulatory nucleic acid molecules, and can thus modify the courseof infection by an intracellular pathogen.

[0050] Various aspects of the invention will now be described in moredetail.

[0051] Nucleic Acid Molecules Comprising Immunomodulatory Nucleic AcidMolecule

[0052] Immunomodulatory nucleic acid molecules are polynucleotides thatmodulate activity of immune cells, especially immune cell activityassociated with a type-1 (Th1-mediated) or type-1 like immune response.Furthermore, immunomodulatory nucleic acid molecules of the presentinvention encompass polynucleotides that inhibit replication ofintracellular pathogens (e.g., inhibit intracellular mycobacterialreplication.).

[0053] Nucleic acid molecules comprising an immunomodulatory nucleicacid molecule which are suitable for use in the methods of the inventioninclude an oligonucleotide, which can be a part of a larger nucleotideconstruct such as a plasmid. The term “polynucleotide” thereforeincludes oligonucleotides, modified oligonucleotides andoligonucleosides, alone or as part of a larger construct. Thepolynucleotide can be single-stranded DNA (ssDNA), double-stranded DNA(dsDNA), single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA). Thepolynucleotide portion can be linearly or circularly configured, or theoligonucleotide portion can contain both linear and circular segments.Immunomodulatory nucleic acid molecules also encompasses crude,detoxified bacterial (e.g., mycobacterial) RNA or DNA, as well asISS-enriched plasmids. “ISS-enriched plasmid” as used herein is meant torefer to a linear or circular plasmid that comprises or is engineered tocomprise a greater number of CpG motifs than normally found in mammalianDNA. Exemplary ISS-enriched plasmids are described in, for example,Roman et al. (1997) Nat Med. 3(8):849-54. Modifications ofoligonucleotides include, but are not limited to, modifications of the3′OH or 5′OH group, modifications of the nucleotide base, modificationsof the sugar component, and modifications of the phosphate group.

[0054] The immunomodulatory nucleic acid molecule can compriseribonucleotides (containing ribose as the only or principal sugarcomponent), deoxyribonucleotides (containing deoxyribose as theprincipal sugar component), or in accordance with the establishedstate-of-the-art, modified sugars or sugar analogs may be incorporatedin the oligonucleotide of the present invention. Examples of a sugarmoiety that can be used include, in addition to ribose and deoxyribose,pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose,lyxose, and a sugar “analog” cyclopentyl group. The sugar may be inpyranosyl or in a furanosyl form. In the modified oligonucleotides ofthe present invention, the sugar moiety is preferably the furanoside ofribose, deoxyribose, arabinose or 2′-O-methylribose, and the sugar maybe attached to the respective heterocyclic bases either in I or Janomeric configuration.

[0055] An immunomodulatory nucleic acid molecule may comprise at leastone nucleoside comprising an L-sugar. The L-sugar may be deoxyribose,ribose, pentose, deoxypentose, hexose, deoxyhexose, glucose, galactose,arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group. TheL-sugar may be in pyranosyl or furanosyl form.

[0056] The phosphorous derivative (or modified phosphate group) that canbe attached to the sugar or sugar analog moiety in the modifiedoligonucleotides of the present invention can be a monophosphate,diphosphate, triphosphate, alkylphosphate, alkanephosphate,phosphoronthioate, phosphorodithioate or the like. The heterocyclicbases, or nucleic acid bases that are incorporated in theoligonucleotide base of the ISS can be the naturally occurring principalpurine and pyrimidine bases, (namely uracil or thymine, cytosine,adenine and guanine, as mentioned above), as well as naturally occurringand synthetic modifications of said principal bases. Those skilled inthe art will recognize that a large number of “synthetic” non-naturalnucleosides comprising various heterocyclic bases and various sugarmoieties (and sugar analogs) are available, and that theimmunomodulatory nucleic acid molecule can include one or severalheterocyclic bases other than the principal five base components ofnaturally occurring nucleic acids. Preferably, however, the heterocyclicbase in the ISS is selected from uracil-5-yl, cytosin-5-yl, adenin-7-yl,adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo [2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrolo [2,3-d] pyrimidin-5-yl,2-amino-4-oxopyrrolo [2,3-d] pyrimidin-3-yl groups, where the purinesare attached to the sugar moiety of the oligonucleotides via the9-position, the pyrimidines via the 1-position, the pyrrolopyrimidinesvia the 7-position and the pyrazolopyrimidines via the 1-position.

[0057] Structurally, the root oligonucleotide of the immunomodulatorynucleic acid molecule is a non-coding sequence that can include at leastone unmethylated CpG motif. The relative position of any CpG sequence inISS with immunomodulatory activity in certain mammalian species (e.g.,rodents) is 5′-CG-3′ (i.e., the C is in the 5′ position with respect tothe G in the 3′ position).

[0058] Immunomodulatory nucleic acid molecules generally do not providefor, nor is there any requirement that they provide for, expression ofany amino acid sequence encoded by the polynucleotide, and thus thesequence of a immunomodulatory nucleic acid molecule may be, andgenerally is, non-coding. Immunomodulatory nucleic acid molecules maycomprise a linear double or single-stranded molecule, a circularmolecule, or can comprise both linear and circular segments.Immunomodulatory nucleic acid molecules may be single-stranded, or maybe completely or partially double-stranded.

[0059] In some embodiments, an immunomodulatory nucleic acid molecule isan oligonucleotide, e.g., consists of a sequence of from about 6 toabout 200, from about 10 to about 100, from about 12 to about 50, orfrom about 15 to about 25, nucleotides in length.

[0060] Exemplary consensus CpG motifs of immunomodulatory nucleic acidmolecules useful in the invention include, but are not necessarilylimited to:

[0061] 5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′, in which theimmunomodulatory nucleic acid molecule comprises a CpG motif flanked byat least two purine nucleotides (e.g., GG, GA, AG, AA, II, etc.,) and atleast two pyrimidine nucleotides (CC, TT, CT, TC, UU, etc.);

[0062] 5′-Purine-TCG-Pyrimidine-Pyrimidine-3′;

[0063] 5′-[TCG]_(n)-3′, where n is any integer that is 1 or greater,e.g., to provide a poly-TCG immunomodulatory nucleic acid molecule(e.g., where n=3, the polynucleotide comprises the sequence5′-TCGTCGTCG-3′); and

[0064] 5′-Purine-Purine-CG-Pyrimidine-Pyrimidine-CG-3′.

[0065] 5′-Purine-TCG-Pyrimidine-Pyrimidine-CG-3′

[0066] Exemplary DNA-based immunomodulatory nucleic acid moleculesuseful in the invention include, but are not necessarily limited to,polynucleotides comprising the following nucleotide sequences: AACGCC,AACGCT, AACGTC, AACGTT; AGCGCC, AGCGCT, AGCGTC, AGCGTT; GACGCC, GACGCT,GACGTC, GACGTT; GGCGCC, GGCGCT, GGCGTC, GGCGTT; ATCGCC, ATCGCT, ATCGTC,ATCGTT; GTCGCC, GTCGCT, GTCGTC, GTCGTT; and TCGTCG, and TCGTCGTCG.

[0067] Octameric sequences are generally the above-mentioned hexamericsequences, with an additional 3′CG. Exemplary DNA-based immunomodulatorynucleic acid molecules useful in the invention include, but are notnecessarily limited to, polynucleotides comprising the followingoctameric nucleotide sequences: AACGCCCG, AACGCTCG, AACGTCCG, AACGTTCG;AGCGCCCG, AGCGCTCG, AGCGTCCG, AGCGTTCG; GACGCCCG, GACGCTCG, GACGTCCG,GACGTTCG; GGCGCCCG, GGCGCTCG, GGCGTCCG, GGCGTTCG; ATCGCCCG, ATCGCTCG,ATCGTCCG, ATCGTTCG; GTCGCCCG, GTCGCTCG, GTCGTCCG, and GTCGTTCG.

[0068] Immunomodulatory nucleic acid molecules useful in the inventioncan comprise one or more of any of the above CpG motifs. For example,immunomodulatory nucleic acid molecules useful in the invention cancomprise a single instance or multiple instances (e.g., 2, 3, 5 or more)of the same CpG motif. Alternatively, the immunomodulatory nucleic acidmolecules can comprises multiple CpG motifs (e.g., 2, 3, 5 or more)where at least two of the multiple CpG motifs have different consensussequences, or where all CpG motifs in the immunomodulatory nucleic acidmolecules have different consensus sequences.

[0069] A non-limiting example of an immunomodulatory nucleic acidmolecule is one with the sequence 5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ IDNO: 1). An example of a control nucleic acid molecule is one having thesequence 5′-TGACTGTGAAgGTTCGAGATGA-3′ (SEQ ID NO:2), which differs fromSEQ ID NO: 1 at the nucleotide indicated in lower case type.

[0070] Immunomodulatory nucleic acid molecules useful in the inventionmay or may not include palindromic regions. If present, a palindrome mayextend only to a CpG motif, if present, in the core hexamer or octamersequence, or may encompass more of the hexamer or octamer sequence aswell as flanking nucleotide sequences.

[0071] The core hexamer structure of the foregoing immunomodulatorynucleic acid molecules can be flanked upstream and/or downstream by anynumber or composition of nucleotides or nucleosides. However, ISS are atleast 6 bases in length, and preferably are between 6 and 200 bases inlength, to enhance uptake of the immunomodulatory nucleic acid moleculeinto target tissues.

[0072] In particular, immunomodulatory nucleic acid molecules useful inthe invention include those that have hexameric nucleotide sequenceshaving “CpG” motifs. Although DNA sequences are generally preferred, RNAimmunomodulatory nucleic acid molecules can be used, with inosine and/oruracil substitutions for nucleotides in the hexamer sequences.

[0073] Modifications

[0074] Immunomodulatory nucleic acid molecules can be modified in avariety of ways. For example, the immunomodulatory nucleic acidmolecules can comprise backbone phosphate group modifications (e.g.,methylphosphonate, phosphorothioate, phosphoroamidate andphosphorodithioate internucleotide linkages), which modifications can,for example, enhance stability of the immunomodulatory nucleic acidmolecule in vivo, making them particularly useful in therapeuticapplications. A particularly useful phosphate group modification is theconversion to the phosphorothioate or phosphorodithioate forms of animmunomodulatory nucleic acid molecule. Phosphorothioates andphosphorodithioates are more resistant to degradation in vivo than theirunmodified oligonucleotide counterparts, increasing the half-lives ofthe immunomodulatory nucleic acid molecules and making them moreavailable to the subject being treated.

[0075] Other modified immunomodulatory nucleic acid moleculesencompassed by the present invention include immunomodulatory nucleicacid molecules having modifications at the 5′ end, the 3′ end, or boththe 5′ and 3′ ends. For example, the 5′ and/or 3′ end can be covalentlyor non-covalently conjugated to a molecule (either nucleic acid,non-nucleic acid, or both) to, for example, increase thebio-availability of the immunomodulatory nucleic acid molecules,increase the efficiency of uptake where desirable, facilitate deliveryto cells of interest, and the like. Exemplary molecules for conjugationto the immunomodulatory nucleic acid molecules include, but are notnecessarily limited to, cholesterol, phospholipids, fatty acids,sterols, oligosaccharides, polypeptides (e.g., immunoglobulins),peptides, antigens (e.g., peptides, small molecules, etc.), linear orcircular nucleic acid molecules (e.g., a plasmid), and the like.Additional immunomodulatory nucleic acid conjugates, and methods formaking same, are known in the art and described in, for example, WO98/16427 and WO 98/55495. Thus, the term “immunomodulatory nucleic acidmolecule” includes conjugates comprising an immunomodulatory nucleicacid molecule.

[0076] Preparation of Immunomodulatory Nucleic Acid Molecules

[0077] Immunomodulatory nucleic acid molecules can be synthesized usingtechniques and nucleic acid synthesis equipment well known in the art(see, e.g., Ausubel et al. Current Protocols in Molecular Biology,(Wiley Intersicence, 1989); Maniatis et al. Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratories, New York, 1982); andU.S. Pat. Nos. 4,458,066; and 4,650,675. Individual polynucleotidefragments can be ligated with a ligase such as T4 DNA or RNA ligase asdescribed in , e.g., U.S. Pat. No. 5,124,246. Oligonucleotidedegradation can be accomplished through exposure to a nuclease, see,e.g., U.S. Pat. No. 4,650,675. As noted above, since theimmunomodulatory nucleic acid molecules need not provide for expressionof any encoded amino acid sequence, the invention does not require thatthe immunomodulatory nucleic acid molecules be operably linked to apromoter or otherwise provide for expression of a coding sequence.

[0078] Alternatively, immunomodulatory nucleic acid molecules can beisolated from microbial species (e.g., mycobacteria) using techniqueswell known in the art such as nucleic acid hybridization, amplification(e.g., by PCR), and the like. Isolated immunomodulatory nucleic acidmolecules can be purified to a substantially pure state, e.g., free ofendogenous contaminants, e.g., lipopolysaccharides. Immunomodulatorynucleic acid molecules isolated as part of a larger polynucleotide canbe reduced to the desired length by techniques well known in the art,such as endonuclease digestion. Other techniques suitable for isolation,purification, and production of polynucleotides to obtain ISS will bereadily apparent to the ordinarily skilled artisan in the relevantfield.

[0079] Circular immunomodulatory nucleic acid molecules can also besynthesized through recombinant methods or chemically synthesized. Wherecircular immunomodulatory nucleic acid molecules are obtained throughisolation or recombinant methods, the immunomodulatory nucleic acidmolecule can be provided as a plasmid. Chemical synthesis of smallercircular oligonucleotides can be performed using methods known in theart (see, e.g., Gao et al. (1995) Nucl. Acids. Res. 23:2025-9; Wang etal., (1994) Nucl. Acids Res. 22:2326-33).

[0080] Where the immunomodulatory nucleic acid molecule comprises amodified oligonucleotide, the modified oligonucleotides can besynthesized using standard chemical techniques. For example,solid-support based construction of methylphosphonates has beendescribed in Agrawal et al. Tet. Lett. 28:3539-42. Synthesis of otherphosphorous-based modified oligonucleotides, such as phosphotriesters(see, e.g., Miller et al. (1971) J. Am Chem Soc. 93:6657-65),phosphoramidates (e.g., Jager et al. (1988) Biochem. 27:7237-46), andphosphorodithioates (e.g., U.S. Pat. No. 5,453,496) is known in the art.Other non-phosphorous-based modified oligonucleotides can also be used(e.g., Stirchak et al. (1989) Nucl. Acids. Res. 17:6129-41).

[0081] Preparation of base-modified nucleosides, and the synthesis ofmodified oligonucleotides using such base-modified nucleosides asprecursors is well known in the art, see, e.g., U.S. Pat. Nos.4,910,300; 4,948,882; and 5,093,232. These base-modified nucleosideshave been designed so that they can be incorporated by chemicalsynthesis into either terminal or internal positions of anoligonucleotide. Nucleosides modified in their sugar moiety have alsobeen described (see, e.g., U.S. Pat. Nos. 4,849,513; 5,015,733;5,118,800; and 5,118,802).

[0082] Techniques for making phosphate group modifications tooligonucleotides are known in the art. Briefly, an intermediatephosphate triester for the target oligonucleotide product is preparedand oxidized to the naturally-occurring phosphate triester with aqueousiodine or other agents, such as anhydrous amines. The resultingoligonucleotide phosphoramidates can be treated with sulfur to yieldphosphorothioates. The same general technique (without the sulfurtreatment step) can be used to produced methylphosphoamidites frommethylphosphonates. Techniques for phosphate group modification are wellknown and are described in, for example, U.S. Pat. Nos. 4,425,732;4,458,066; 5,218,103; and 5,453,496.

[0083] Identification of Immunomodulatory Nucleic Acid Molecules

[0084] Confirmation that a particular compound has the properties of animmunomodulatory nucleic acid molecule useful in the invention can beobtained by evaluating whether the immunomodulatory nucleic acidmolecule elicits the appropriate cytokine secretion patterns, e.g., acytokine secretion pattern associated with a type-i imrnune response;inhibits intracellular pathogen replication, e.g., inhibitsintracellular growth of intracellular pathogens either in vitro or invivo; and/or modulates intracellular availability of cellular productsnecessary for growth and/or reproduction of the intracellular pathogen,e.g., reduces intracellular levels of L-tryptophan, for example, byinducing expression of indoleamine 2,3-dioxygenase (IDO) in a cell. ISSdelivered with an antigen also induces activity of cytotoxic T cells andacts as a very strong mucosal adjuvant (see, e.g., Horner (1998) Cell.Immunol. 190:77-82). As noted above, immunomodulatory nucleic acidmolecules of interest in the methods of the invention are those thatelicit a Th1-mediated response, those that induce expression of IDO, andthose that inhibit intracellular growth of intracellular pathogens,particularly intracellular growth of mycobacteria, more particularlyintracellular mycobacterial growth in macrophages, especiallymonocyte-derived macrophages and bone marrow-derived macrophages.

[0085] In general, helper T (Th) cells are divided into broad groupsbased on their specific profiles of cytokine production: Th1, Th2, andThO. “Th1 ” cells are T lymphocytes that release predominantly thecytokines IL-2 and IFN-γ, which cytokines in turn promote T cellproliferation, facilitate macrophage activation, and enhance thecytolytic activity of natural killer (NK) cells and antigen-specificcytotoxic T cells (CTL). In contrast, the cytokines predominantlyreleased by Th2 cells are IL-4, IL-5, and IL-10. IL-4 and IL-5 are knownto mediate antibody isotype switching towards IgE or IgA response, andpromote eosinophil recruitment, skewing the immune system toward an“allergic” type of response. ThO cells release a set of cytokines withcharacteristics of both Th1-type and Th2-type responses. While thecategorization of T cells as Th1, TH2, or Th0 is helpful in describingthe differences in immune response, it should be understood that it ismore accurate to view the T cells and the responses they mediate asforming a continuum, with Th1 and Th2 cells at opposite ends of thescale, and ThO cells providing the middle of the spectrum. Therefore, itshould be understood that the use of these terms herein is only todescribe the predominant nature of the immune response elicited, and isnot meant to be limiting to an immune response that is only of the typeindicated. Thus, for example, reference to a “type-1” or “Th1” immuneresponse is not meant to exclude the presence of a “type-2” or “Th2”immune response, and vice versa.

[0086] Details of in vitro and in vivo techniques useful for evaluationof production of cytokines associated with a type-1 or type2 response,as well as for evaluation of antibody production, are well known in theart. Likewise, methods for evaluating the ability of candidate ISS toinhibit intracellular pathogen growth are also well known in the art,and are further exemplified in the Examples below.

[0087] Administration of Immunomodulatory Nucleic Acid Molecules

[0088] Immunomodulatory nucleic acid molecules are administered to anindividual using any available method and route suitable for drugdelivery, including in vivo and ex vivo methods, as well as systemic,mucosal, and localized routes of administration.

[0089] Conventional and pharmaceutically acceptable routes ofadministration include intranasal, intramuscular, intratracheal,subcutaneous, intradermal, topical application, intravenous, rectal,nasal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe immunomodulatory nucleic acid molecule and/or the desired effect onthe immune response. The immunomodulatory nucleic acid composition canbe administered in a single dose or in multiple doses, and may encompassadministration of booster doses, to elicit and/or maintain the desiredeffect on the immune response.

[0090] Immunomodulatory nucleic acid molecules can be administered to asubject using any available conventional methods and routes suitable fordelivery of conventional drugs, including systemic or localized routes.Methods and localized routes that further facilitate production of atype-1 or type-1-like response and/or the anti-pathogenic (e.g.anti-mycobacterial) activity of the immunomodulatory nucleic acidmolecules, particularly at or near a site of intracellular pathogeninfection (e.g., within the lungs) is of interest in the invention, andmay be preferred over systemic routes of administration, both for theimmediacy of therapeutic effect and avoidance of in vivo degradation ofthe administered immunomodulatory nucleic acid molecules. In general,routes of administration contemplated by the invention include, but arenot necessarily limited to, enteral, parenteral, or inhalational routes.Inhalational routes may be preferred in cases of pulmonary involvement,particularly in view of the activity of immunomodulatory nucleic acidmolecules as a mucosal adjuvant.

[0091] Inhalational routes of administration (e.g., intranasal,intrapulmonary, and the like) are particularly useful in stimulating animmune response for prevention or treatment of intracellular pathogeninfections of the respiratory tract. Such means include inhalation ofaerosol suspensions or insufflation of the polynucleotide compositionsof the invention. Nebulizer devices, metered dose inhalers, and the likesuitable for delivery of polynucleotide compositions to the nasalmucosa, trachea and bronchioli are well-known in the art and willtherefore not be described in detail here. For. general review in regardto intranasal drug delivery, see, e.g., Chien, Novel Drug DeliverySystems, Ch. 5 (Marcel Dekker, 1992).

[0092] Parenteral routes of administration other than inhalationadministration include, but are not necessarily limited to, topical,transdermal, subcutaneous, intramuscular, intraorbital, intracapsular,intraspinal, intrasternal, and intravenous routes, i.e., any route ofadministration other than through the alimentary canal. Parenteraladministration can be carried to effect systemic or local delivery ofimmunomodulatory nucleic acid molecules. Where systemic delivery isdesired, administration typically involves invasive or systemicallyabsorbed topical or mucosal administration of pharmaceuticalpreparations.

[0093] Immunomodulatory nucleic acid molecules can also be delivered tothe subject by enteral administration. Enteral routes of administrationinclude, but are not necessarily limited to, oral and rectal (e.g.,using a suppository) delivery.

[0094] Methods of administration of immunomodulatory nucleic acidmolecules through the skin or mucosa include, but are not necessarilylimited to, topical application of a suitable pharmaceuticalpreparation, transdermal transmission, injection and epidermaladministration. For transdermal transmission, absorption promoters oriontophoresis are suitable methods. For review regarding such methods,those of ordinary skill in the art may wish to consult Chien, supra atCh. 7. lontophoretic transmission may be accomplished using commerciallyavailable “patches” which deliver their product continuously viaelectric pulses through unbroken skin for periods of several days ormore. An exemplary patch product for use in this method is the LECTROPATCH™ (manufactured by General Medical Company, Los Angeles, Calif.)which electronically maintains reservoir electrodes at neutral pH andcan be adapted to provide dosages of differing concentrations, to dosecontinuously and/or to dose periodically.

[0095] Epidermal administration can be accomplished by mechanically orchemically irritating the outermost layer of the epidermis sufficientlyto provoke an immune response to the irritant. An exemplary device foruse in epidermal administration employs a multiplicity of very narrowdiameter, short tynes which can be used to scratch ISS coated onto thetynes into the skin. The device included in the MONO-VACC™ tuberculintest (manufactured by Pasteur Merieux, Lyon, France) is suitable for usein epidermal administration of immunomodulatory nucleic acid molecules.

[0096] The invention also contemplates opthalmic administration ofimmunomodulatory nucleic acid molecules, which generally involvesinvasive or topical application of a pharmaceutical preparation to theeye. Eye drops, topical cremes and injectable liquids are all examplesof suitable formulations for delivering drugs to the eye.

[0097] Immunomodulatory nucleic acid molecules can be administered to asubject prior to exposure to intracellular pathogen, after exposure tointracellular pathogen but prior to onset of disease symptoms associatedwith infection, or after intracellular pathogen infection or onset ofdisease symptoms. As such, immunomodulatory nucleic acids can beadministered at any time after exposure to intracellular pathogen, but afirst dose is usually administered about 8 hours, about 12 hours, about24 hours, about 2 days, about 4 days, about 8 days, about 16 days, about30 days or 1 month, about 2 months, about 4 months, about 8 months, orabout 1 year after exposure to intracellular pathogen. As described inmore detail below, the invention also provides for administration ofsubsequent doses of immunomodulatory nucleic acid molecules.

[0098] Administration With Additional Chemotherapeutic Agents

[0099] In one embodiment, immunomodulatory nucleic acid molecules areadministered in combination with a conventional anti-pathogenic agent toprovide for a synergistic effect in treatment of intracellular pathogeninfection. The additional anti-pathogenic agent may be any agent (e.g.,chemotherapeutic agent) identified as having activity against theintracellular pathogen of interest (e.g., in inhibition of extracellularor intracellular growth stages of the intracellular pathogen (e.g.,mycobacteria), enhancement of intracellular pathogen clearance (e.g.,mycobacteria), etc.). Exemplary anti-pathogenic agents include, but arenot necessarily limited to, antibiotics, including antimicrobial agents,(e.g., bacteriostatic and bacteriocidal agents (e.g., aminoglycosides,β-lactam antibiotics, cephalosporins, macrolides, penicillins,tetracyclines, quinolones, and the like ), antivirals (e.g.,amprenavirs, acyclovirs, amantadines, virus penciclovirs, and the like),and the like), antifungals, (e.g., imidazoles, triazoles, allylamines,polyenes, and the like), as well as anti-parasitic agents (e.g.,atovaquones, chloroquines, pyrimethamines, ivermectins, mefloquines,pentamidines, primaquines, and the like). Where the subject beingtreated is particularly susceptible to infection by intracellularpathogens, including opportunistic pathogens, it may be desirable toadminister immunomodulatory nucleic acid molecules in a combinationtherapeutic regimen with chemotherapeutic agents that exhibit activityagainst microbial and/or parasitic pathogens, e.g., antimicrobialagents, antiviral agents, antifungal agents, anti-parasitic agents, etc.Such combination therapies can involve simultaneous or consecutiveadministration of ISS and such a chemotherapeutic agent(s).

[0100] Specific exemplary conventional anti-pathogenic/chemotherapeuticagents and combinatory therapies, particularly anti-mycobacterial agentsand combinatory therapies, include, but are not necessarily limited to,clarithromycin (e.g., by oral administration or injection); capreomycinsulfate (e.g., by intramuscular injection or intravenous infusion, e.g.CAPASTAT®); ethambutol HCl (e.g., by oral administration of tablets orcapsules, e.g., MYAMBUTOL®); isoniazid (e.g., by intramuscular injectionor oral administration, e.g., NYDRAZID®; aminosalicylic acid (e.g.,aminosalicyclic acid granules for oral administration, e.g.,PASER®GRANULES); rifapentine (e.g., by oral administration; e.g.,PRIFTIN®); PYRAZINAMIDE (e.g., by oral administration); rifampin (e.g.,by oral administration, e.g., RIFADIN®, or by intravenousadministration, e.g., RIFADIN IV®); rifampin and isoniazid combinationtherapy (e.g., by oral administration, e.g., RIFAMATE®); rifampin,isoniazid, and pyrazinamide combination therapy (e.g., by oraladministration, e.g., RIFATER®); cycloserine (e.g., by oraladministration, e.g., SEROMYCIN®; streptomycin sulfate (e.g., byinjection or oral administration); ethionamide (e.g., by oraladministration, e.g., TRECATOR®-SC), and the like.

[0101] The anti-pathogenic/chemotherapeutic agent and immunomodulatorynucleic acid molecule can be administered within the same or differentformulation; by the same or different routes; or concurrently,simultaneously, or consecutively. The immunomodulatory nucleic acidmolecule can be delivered according to a regimen (e.g., frequency duringa selected interval (e.g., number of times per day), delivery route,etc.) that is the same as, similar to, or different from that of theanti-pathogenic agent. When administered in combination, ISS and ananti-pathogenic agent are generally administered within about 96 hours,about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 8hours, about 4 hours, about 2 hours, about 1 hour, or about 30 minutesor less, of each other. Thus, although it may be desirable to do so insome situations, it is not necessarily required that ISS and ananti-pathogenic agent (e.g., antibacterial agent) be deliveredsimultaneously.

[0102] Dosages

[0103] One particular advantage of the use of immunomodulatory nucleicacid molecules in the methods of the invention is. that immunomodulatorynucleic acid molecules exert immunomodulatory and anti-pathogenicactivity even at relatively low dosages. Although the dosage used willvary depending on the clinical goals to be achieved, a suitable dosagerange is one which provides up to about 1 μg, to about 1,000 μg, toabout 10,000 μg, to about 25,000 μg or about 50,000 μg of ISS.Immunomodulatory nucleic acid molecules can be administered in a singledosage or several smaller dosages over time. Alternatively, a targetdosage of ISS can be considered to be about 1-10,uM in a sample of hostblood drawn within the first 24-48 hours after administration of ISS.Based on current studies, immunomodulatory nucleic acid molecules arebelieved to have little or no toxicity at these dosage levels.

[0104] It should be noted that the immunotherapeutic activity ofimmunomodulatory nucleic acid molecules in the invention is essentiallydose-dependent. Therefore, to increase ISS potency by a magnitude oftwo, each single dose is doubled in concentration. Increased dosages maybe needed to achieve the desired therapeutic goal. The invention thuscontemplates administration of “booster” doses to provide and maintainan immune response effective to protect the subject from infection or toinhibit infection; to reduce the risk of the onset of disease or theseverity of disease symptoms that may occur as a result of infection; tofacilitate reduction of intracellular pathogen load; and/or tofacilitate clearance of infecting intracellular pathogen from thesubject (e.g., to facilitate clearance of organisms from the lungs).When multiple doses are administered, subsequent doses are administeredwithin-about 16 weeks, about 12 weeks, about 8 weeks, about 6 weeks,about 4 weeks, about 2 weeks, about 1 week, about 5 days, about 72hours, about 48 hours, about 24 hours, about 12 hours, about 8 hours,about 4 hours, or about 2 hours or less of the previous dose. In oneembodiment, ISS are administered at intervals ranging from at leastevery two weeks to every four weeks (e.g., monthly intervals) in orderto maintain the maximal immune response against intracellular pathogeninfection (e.g., mycobacterial infection).

[0105] In view of the teaching provided by this disclosure, those ofordinary skill in the clinical arts will be familiar with, or canreadily ascertain, suitable parameters for administration of ISSaccording to the invention.

[0106] Formulations

[0107] In general, immunomodulatory nucleic acid molecules are preparedin a pharmaceutically acceptable composition for delivery to a host.Pharmaceutically acceptable carriers preferred for use with the ISS ofthe invention may include sterile aqueous of non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. A composition of ISS may also belyophilized using means well known in the art, for subsequentreconstitution and use according to the invention. Also of interest areformulations for liposomal delivery, and formulations comprisingmicroencapsulated immunomodulatory nucleic acid molecules.

[0108] In general, the pharmaceutical compositions can be prepared invarious forms, such as granules, tablets, pills, suppositories,capsules, suspensions, salves, lotions and the like. Pharmaceuticalgrade organic or inorganic carriers and/or diluents suitable for oraland topical use can be used to make up compositions comprising thetherapeutically-active compounds. Diluents known to the art includeaqueous media, vegetable and animal oils and fats. Stabilizing agents,wetting and emulsifying agents, salts for varying the osmotic pressureor buffers for securing an adequate pH value, and skin penetrationenhancers can be used as auxiliary agents. Preservatives and otheradditives may also be present such as, for example, anti-pathogenicagents (e.g., antimicrobials, antibacterials, antivirals, antifungals,etc.), antioxidants, chelating agents, and inert gases and the like. Inone embodiment, as discussed above, the immunomodulatory nucleic acidmolecule formulation comprises an additional anti-pathogenic agent.Exemplary anti-pathogenic agents include, but are not necessarilylimited to, antibiotics, including antimicrobial agents (e.g.,bacteriostatic and bacteriocidal agents (e.g., aminoglycosides, β-lactamantibiotics, cephalosporins, macrolides, penicillins, tetracyclines,quinolones, and the like ), antivirals (e.g., amprenavirs, acyclovirs,amantadines, virus penciclovirs, and the like), and the like),antifungals, (e.g., imidazoles, triazoles, allylamines, polyenes, andthe like), as well as anti-parasitic agents (e.g., atovaquones,chloroquines, pyrimethamines, ivermectins, mefloquines, pentamidines,primaquines, and the like). In another embodiment, the anti-pathogenicagent is an anti-mycobacterial agent (e.g., clarithromycin; capreomycinsulfate; ethambutol HCl; isoniazid; aminosalicylic acid; rifapentine;PYRAZINAMIDE; rifampin; rifampin and isoniazid in combination; rifampin,isoniazid, and pyrazinamide in combination; cycloserine; streptomycinsulfate; ethionamide; and the like).

[0109] Immunomodulatory nucleic acid molecules can be administered inthe absence of agents or compounds that might facilitate uptake bytarget cells (e.g., as a “naked” polynucleotide, e.g., a polynucleotidethat is not encapsulated by a viral particle). Immunomodulatory nucleicacid molecules can also be administered with compounds that facilitateuptake of immunomodulatory nucleic acid molecules by target cells (e.g.,by macrophages) or otherwise enhance transport of the immunomodulatorynucleic acid molecules to a treatment site for action. Absorptionpromoters, detergents and chemical irritants (e.g., keratinolyticagents) can enhance transmission of an immunomodulatory nucleic acidmolecule composition into a target tissue (e.g., through the skin). Forgeneral principles regarding absorption promoters and detergents whichhave been used with success in mucosal delivery of organic andpeptide-based drugs, see, e.g., Chien, Novel Drug Delivery Systems, Ch.4 (Marcel Dekker, 1992). Examples of suitable nasal absorption promotersin particular are set forth at Chien, supra at Ch. 5, Tables 2 and 3;milder agents are preferred. Suitable agents for use in the method ofthis invention for mucosal/nasal delivery are also described in Chang,et al., Nasal Drug Delivery, “Treatise on Controlled Drug Delivery”, Ch.9 and Tables 3-4B thereof, (Marcel Dekker, 1992). Suitable agents whichare known to enhance absorption of drugs through skin are described inSloan, Use of Solubility Parameters from Regular Solution Theory toDescribe Partitioning-Driven Processes, Ch. 5, “Prodrugs: Topical andOcular Drug Delivery” (Marcel Dekker, 1992), and at places elsewhere inthe text. All of these references are incorporated herein for the solepurpose of illustrating the level of knowledge and skill in the artconcerning drug delivery techniques.

[0110] A colloidal dispersion system may be used for targeted deliveryof immunomodulatory nucleic acid molecules to specific tissue. Colloidaldispersion systems include macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes.

[0111] Liposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 μm canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA and DNA can be encapsulated within the aqueousinterior and be delivered to cells in a biologically active form(Fraley, et al., (1981) Trends Biochem. Sci., 6:77). The composition ofthe liposome is usually a combination of phospholipids, particularlyhigh-phase-transition-temperature phospholipids, usually in combinationwith steroids, especially cholesterol. Other phospholipids or otherlipids may also be used. The physical characteristics of liposomesdepend on pH, ionic strength, and the presence of divalent cations.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0112] Where desired, targeting of liposomes can be classified based onanatomical and mechanistic factors. Anatomical classification is basedon the level of selectivity, for example, organ-specific, cell-specific,and organelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0113] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various well known linking groups can be used forjoining the lipid chains to the targeting ligand (see, e.g., Yanagawa,et al., (1988) Nuc. Acids Symp. Ser., 19:189; Grabarek, et al., (1990)Anal. Biochem., 185:131; Staros, et al., (1986) Anal. Biochem. 156:220and Boujrad, et al., (1993) Proc. Natl. Acad. Sci. USA, 90:5728).Targeted delivery of immunomodulatory nucleic acid molecules can also beachieved by conjugation of the ISS to the surface of viral and non-viralrecombinant expression vectors, to an antigen or other ligand, to amonoclonal antibody or to any molecule which has the desired bindingspecificity.

[0114] Additional Formulation Components

[0115] In addition to immunomodulatory nucleic acid molecules, theformulations suitable for treatment or prevention of intracellularpathogen infections according to the present invention can compriseactive or inactive components in lieu of or in addition to thecomponents described above. For example, the formulation may compriseanti-pathogenic agents (e.g., antibiotics), particularly where the ISSis administered for treatment of an active infection. In one embodiment,the immunomodulatory nucleic acid molecule is administered with arelevant antigen to further enhance the subject's immune responseagainst one or more species of intracellular pathogen. In anotherembodiment, the immunomodulatory nucleic acid molecule is administeredwith one or more mycobacterial antigens. Mycobacterial antigens ofinterest may include, but are not necessarily limited to, the 65 kDaantigen and antigen 85B of M. avium (Velaz-Faircloth, et al. (1999)Infect. Immun. 67:4243-4250); the antigen 85B, ESAT-6 and MPT64 of M.tuberculosis (Kamath, et al. (1999) Infect. Immun. 67:1702-1707), and M.tuberculosis hsp-65 (Bonato, et al. (1998) Infect. Immun. 66:169-175).

[0116] Kits

[0117] The present invention also provides kits for use in the methodsdescribed herein. Such kits may include any or all of the following: 1)ISS; 2) a pharmaceutically acceptable carrier (which may be pre-mixedwith the ISS) or suspension base for reconstituting lyophilized ISS; 3)additional medicaments; 4) a sterile vial for each ISS and additionalmedicament, or a single vial for mixtures thereof; 5) device(s) for usein delivering ISS to a host; 6) assay reagents for detecting indiciathat the desired immunomodulatory effects have been accomplished in thesubject to which the ISS has been administered and a suitable assaydevice.

[0118] Intracellular Pathogen Infections Amenable to Treatment

[0119] The methods and compositions described herein can be used in thetreatment or prevention of any of a variety of infections byintracellular pathogens (e.g., viruses, bacteria, protozoa, fungi, andintracellular parasites) in a variety of subjects susceptible to orhaving such infections. In one embodiment, the intracellular pathogeninfection is a mycobacterial infection. Of particular interest is thetreatment and/or prevention of infection or disease by M tuberculosis,M. avium (or M. avium-intracellulare), M. leprae (particularly M. lepraeinfection leading to tuberculoid leprosy), M. kansasii, M. fortuitum, M.chelonae, and M. absecessus. While treatment of humans is of particularinterest, the methods of the invention can also be used to preventintracellular pathogen infection or disease in non-human subjects. Forexample, M. avium causes lymphadenitis in slaughter pigs; M.paratuberculosis infection causes paratuberculosis, a tuberculosis-likedisease that can result in great production losses in cattle, sheep andgoats; and M. bovis is carried by cattle and can cause a tuberculin-likeinfection in humans.

[0120] Immunomodulatory nucleic acid molecules can be administeredprophylactically or following onset of disease. Prophylactic therapy caninvolve administration of immunomodulatory nucleic acid molecules priorto exposure to intracellular pathogen, or can be after exposure, butprior to establishment of infection or disease (e.g., the subject may becolonized by intracellular pathogen, but not exhibit or yet exhibitsymptoms associated with disease caused by the intracellular pathogendue to the subject being a carrier or having been exposed to asub-infectious dose).

[0121] The methods and compositions of the invention may be particularlyadvantageous in the treatment of infection by drug-resistant strains ofintracellular pathogen, as well as treatment of intracellular pathogeninfections in immunocompromised hosts.

EXAMPLES

[0122] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

[0123] Methods and Materials

[0124] The following procedures are used in the Examples described indetail below. Although some of the methods described below are in commonuse, the specific protocol used in the Examples below is described indetail where alternative protocols are often employed. Basic proceduressuch as DNA digestion by restriction enzymes and ligation are notdescribed, as such are well within the skill of the ordinarily skilledartisan and, in some instances, are carried out according to the enzymeor kit manufacturer's instructions.

[0125] Mice. Female C57B1/6 mice (7 to 8 week-old) were purchased fromThe Jackson Laboratory (Bar Harbor, Me.) and from Charles RiverLaboratories, Inc. (Wilmington, M. AVIUM). Female 129/SvEv mice werepurchased from Taconic Laboratories, Germantown, N.Y. The induciblenitric oxide synthetase (iNOS)^(−/−), IL-12 p40^(−/−), TNF-α^(−/−), andNADPH oxidase (gp91phox)^(−/−) knockout mice are on the C57B1/6background, and were purchased from The Jackson Laboratory. IFN-α/βreceptor (IFN-α/βR)^(−/−) and IFN-γ receptor (IFN-γR)^(−/−) (129/EvSvbackground) knockout mice were obtained from B & K Universal Ltd. (EastYorkshire, United Kingdom).

[0126] Reagents and cytokines. Endotoxin-free (<1 ng/mg DNA),phosphorothioate single-stranded oligodeoxynucleotides were obtainedfrom Trilink Biotechnologies, San Diego, Calif. The sequence of the ISSwas 5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ ID NO: 1). The sequence of themutated-ODN (M-ODN) was 5′-TGACTGTGAAGGTTAGAGATGA-3′ (SEQ ID NO:3). Theunderlined bases indicate the CpG motif of the polynucleotide or itscorresponding 2 base alteration.

[0127] Unless otherwise noted, 10 μg/ml ISS or M-ODN was used.L-tryptophan (L-try) was obtained from Gibco BRL (Grand Island, N.Y.)and DMEM was supplemented with L-try for a final concentration of 66μg/ml. 1-methyl-DL-tryptophan (M-try) was purchased from AldrichChemicals (Milwaukee, Wis.). Anti-CD3, anti-CD28, anti-CD4, and anti-CD8monoclonal antibodies as well as monensin and murine recombinant IFN-γwere purchased from BD Pharmingen (San Diego, Calif.). AbbottLaboratories (Abbott Park, Ill.) kindly provided Clarithromycin (CLA).

[0128] Culture of M. avium. A previously studied M. avium strain 13(Hayashi, et al. (1999) Infect. Immun. 67:3558-3565; Meylan, et al.(1990) Infect. Immun. 58:2564-2568), isolated from an AIDS patient atUCSD, San Diego, Calif., was used in all experiments. The organism wascultured on Middlebrook 7H 11 agar (Difco Laboratories, Detroit, Mich.)with OADC enrichment at 37° C. in the presence of 5% CO₂ for two weeks.Transparent colonies were selectively picked and further cultured onMiddlebrook 7H 11 plates for two more weeks. The resulting colonies,which were predominantly transparent (>90%), were then collected andwashed two times with phosphate-buffered saline (PBS). The bacteria werefinally resuspended in Middlebrook 7H9 broth (Difco Laboratories) andthe OD₆₀₀ of the suspension was adjusted to 0.15-0.2. The suspension wasaliquoted and stored at −70° C. until use. The number of organisms/ml ofthis suspension was determined by the colony forming unit (CFU) assay.

[0129] CFU assay. The number of CFU in a given sample was determined bycolony counting as described previously (Ogata, et al. (1992) Infect.Immun. 60:4720-4725). Serial 10-fold dilutions of total cell lysates ortissue homogenates were performed in PBS. 10 μl of each dilution wereplated on Middlebrook 7H 11 plates supplemented with OADC enrichment.The plates were incubated up to 14 days at 37° C. The number of colonieswas counted every alternate day from day 10 onwards until no newcolonies appeared. This yielded the number of CFU/10 μl. The number ofCFU in each well was then calculated.

[0130] Isolation of human monocytes. Monocytes were isolated from normalhuman buffy coats obtained from the San Diego Blood Bank byFicoll-Hypaque and Percoll gradient centrifugation (Hayashi, et al.(1997) Infect. Immun. 65:5262-5271). Purity of the monocytes by thismethod was greater than 70%. The monocytes thus isolated were culturedfor five to seven days in Iscove's Modified Dulbecco's Medium(BioWhittaker, Walkersville, Md.) supplemented with 10% normal humanserum (Irvine Scientific, Santa Ana, Calif.), 2 mM L-glutamine and 50units/ml penicillin-streptomycin in Teflon beakers to yield humanmonocyte-derived macrophages (hMDM). These hMDM were further enriched byadherence to the wells of tissue culture plates before use inexperiments. Purity of hMDM after the second adherence assessed byesterase staining was >95%. Viability determined by trypan blueexclusion was >97%.

[0131] Preparation of murine macrophages. Mouse bone marrow-derivedmacrophages (mBMDM) were prepared from mouse bone marrow using L-cellconditioned media, as described in Martin-Orozco, et al. (1999)Immunology 11: 1111-1118.

[0132] Effect of ISS on intracellular growth of M. avium in vitro. hMDM(6×10⁴) were adhered to the wells of 96-well tissue culture plates fortwo hours and non-adherent cells were removed by washing with warmRPMI-1640. Adherent cells were treated with ISS at 3, 10, or 30 μg/mlfor 72 h before infection with M. avium. Cells treated with M-ODN at 10μg/ml or with media alone served as controls. After 72 h, the cells werewashed two times and infected with M. avium in RPMI-1640 supplementedwith 5% heat-inactivated fetal bovine serum (FBS, Irvine Scientific) ata cell:bacteria ratio of 1:5-1: 10. On days 1, 3 and 7 after infection,the total number of colony forming units (CFU) in each well wasdetermined. The supernatant was collected and set aside and the adherentcells were lysed. To account for all the mycobacteria in each well,corresponding lysates and supernatants were combined and used todetermine the number of CFU. Experiments were done in triplicate andresults were expressed as mean±SD of CFU per well.

[0133] Alternatively, 5×10⁴ mBMDM or hMDM was treated with ISS for 3days before infection. Macrophages treated with M-ODN or with mediaalone served as controls. After 3 days, the cells were infected for 2hrs with M. avium at a macrophage:bacteria ratio of 2:1 for mBMDM or of1:10 for hMDM, and subsequently cultured in fresh media withoutantibiotics. To examine attachment and/or invasion of M. avium, themacrophages were lysed immediately after washing, and the number ofbacteria were enumerated by the CFU assay. Intracellular growth of M.avium was determined on days 1, 3, and 7 after infection. To account forall the mycobacteria in each group, corresponding lysates were combinedand then the number of CFU was determined. To examine the efficacy ofanti-mycobacterial treatments, CFU recovered from cells treated withmedium alone were considered as 100% growth.

[0134] To determine whether treatment with ISS after infection alters M.avium growth, adherent cells were first infected with M. avium for 2 hrsand then these cells were cultured with fresh media containing ISS.Infected cells treated with M-ODN or medium alone served as controls. Atday 7, intracellular growth of M. avium was assessed by CFU assay.

[0135] Effect of ISS on growth of M. avium in vivo in mice. Three daysbefore infection, the experimental mice (n=5) were injectedintradermally with ISS (100 μg/mouse in 50 μl). The control mice (n=5)received PBS (50 I11/mouse). All mice were infected intravenously withM. avium (1×10⁶/mouse or 1×10⁷/mouse). At 2, 4, and 6 weeks afterinfection, mice were sacrificed and the spleen, liver, and lungs fromeach mouse were collected and weighed. Blood was collected by cardiacpuncture and used in the ELISA studies described below. A section ofeach organ was minced and homogenized for 30 s with 0.25% SDS in PBS (1ml/100 mg of tissue). The number of CFU in the tissue homogenates wasdetermined by the CFU assay and the results were expressed as CFU/organ.All procedures were performed under a biosafety cabinet in a biosafetylevel 2 facility.

[0136] To study the effect of ISS when combined with CLA in vivo, 25mice were injected with M. avium (1×10⁷/mouse). One week afterinfection, treatment with either ISS or M-ODN and CLA was initiated. Themice were divided into 5 treatment groups (n=5 per group): group 1, notreatment; group 2, ISS alone; group 3, CLA alone; group 4, CLA and ISS;and group 5, CLA and M-ODN. CLA (200 mg/kg) was administeredintraperitoneally three times a week for four weeks (Doherty, et al.(1998) J. Immunol. 160:5428-5435) and bacterial growth in the spleen,liver and lungs was determined.

[0137] Intracellular IFN-γ staining and detection of secreted IFN-γ byELISA. Mice were injected intradermally (i.d.) with ISS (50 μg/mouse).The control mice received M-ODN (50 μg/mouse) or PBS (50 μl/mouse). Allmice were infected with 1×10⁷ M. avium. Three weeks after infection, themice were sacrificed and splenocytes from mice receiving the sametreatment were pooled. Intracellular cytokine staining was performedusing the Cytofix/Cytoperm kit (Pharmingen) according to themanufacturer's instructions. Briefly, the splenocytes were stimulatedwith anti-CD3 and anti-CD28 activating antibodies in the presence ofmonensin to allow intracellular IFN-γ to accumulate for 6 hrs. Next,surface CD4 and CD8 were stained for, the cells were fixed, and theplasma membranes were permeabilized, allowing for intracellular stainingwith anti-IFN-γ. The cells were analyzed on a FACSCalibur flow cytometer(Becton Dickenson). To study IFN-γ production, splenocytes wereincubated with anti-CD3 and anti-CD28 antibodies in vitro for 24 hrs,and these supernatants were assayed for the presence of IFN-γ bysandwich ELISA (Martin-Orozco, et al. (1999) Int. Immun. 11:1111-1118).

[0138] RNA extraction, RT-PCR, and IDO activity assay. 1-4×10⁶ mBMDMwere treated with ISS or M-ODN. After 3 days, the cells were infectedwith M. avium for 2 hrs. At 2, 24, and 48 hrs after infection, the M.avium-infected macrophages were lysed and total RNA was isolated usingthe Trizol Reagent (Gibco BRL). The induction of IDO gene transcriptionwas measured by semi-quantitative RT-PCR. First-strand cDNA preparationand PCR amplification was carried out using the SuperScriptPre-amplification System (Gibco BRL) and AdvanTaq Plus DNA polymerase(Clontech, San Francisco, Calif.), respectively. PCR products werevisualized by electrophoresis on 2% agarose gels. The primer sequencesused were as follows: IDO: 5′-TTATGCAGACTGTGTCCTGGCAAA-3′ and5′-TTTCCAGCCAGACAGATATATGCG-3′, G3PDH: 5′-ACCACAGTCCATGCCATCAC-3′ and5′-TCCACCACCCTGTTGCTGTA-3′

[0139] IFN-γ and IL-12 measurement in the serum of M. avium-infectedmice. Serum obtained from M. avium-infected mice that received ISS orPBS was assayed for IFN-γ and IL-12 by ELISA using mouse IFN-γ and IL-12(p70) ELISA kits (Endogen, Inc. Woburn, M. AVIUM), respectively, andfollowing the manufacturer's instructions.

[0140] Histologic examination. Sections of the spleen and livercollected from the experimental and control M. avium-infected mice at 2,4, and 6 weeks post-infection, were fixed overnight in 10% bufferedformalin at room temperature, and embedded in paraffin. Theparaffin-embedded tissue was further sectioned (5 μm thickness), stainedwith hematoxylin-eosin and observed under an Olympus microscope. Atleast three sections from each organ of each of the experimental andcontrol mice were evaluated and representative fields were viewed at100×.

[0141] Statistical analysis. Results were expressed as mean±SD.Statistical differences were determined using the Student's t test(two-tailed distribution). A P value at or below 0.05 was considered tobe statistically significant.

Example 1 In Vitro Effect of ISS on the Growth of M. avium in Human MDM

[0142] To examine whether ISS has a direct effect on the growth of M.avium in human monocyte-derived macrophages (hMDM), hMDM were preparedas described above (6×10⁴ hMDM/well) and pretreated with ISS or M-ODNfor 72 h before infection and then infected with M. avium. The number ofCFU within the cells was determined on days 1, 3 and 7 post-infection.

[0143] On day 7, maximum inhibition of intracellular growth of M. aviumwas observed in the case of hMDM pretreated with 3 μg/ml of ISS(91.3±1.7%) in comparison with HMDM treated with medium alone or thosetreated with M-ODN (p<0.001) (FIG. 1). There was no further increase ingrowth inhibition at the higher concentrations of ISS tested. Theseresults demonstrate that ISS can directly stimulate macrophages torestrict the intracellular growth of M. avium.

[0144] In summary, these data show that pretreatment of hMDM with ISSsignificantly inhibited the intracellular growth of M. avium for up to 7days, demonstrating that ISS may directly activate macrophages to killM. avium.

Example 2 Effect of Treatment with ISS Upon Infected hMDM

[0145] To study the effect of ISS upon growth of M. avium in an on-goinginfection of hMDM, hMDM were infected as described above (6×10⁴hMDM/well) and incubated with ISS (10 mg/ml) either immediately afterinfection, or one day after infection. In some wells hMDM werepretreated with ISS (10 mg/ml) three days before infection and were theninfected with M. avium as described above. CFU of these wells wasdetermined at day 7. Experiments were done in triplicate and resultswere expressed as mean±SD of CFU per well.

[0146] Pretreatment of ISS three days before infection (-3 days)inhibited intracellular growth of M. avium 51±4% compared to mediumalone as the control (FIG. 2). Treatment with ISS immediately afterinfection (0 day) and one day after infection (+1 day) inhibited growthof M. avium 53±18% and 36+16%, respectively. These data indicate thattreatment with ISS after infection can also activate hMDM to inhibit M.avium growth as well as pretreatment with ISS.

Example 3 Effect of ISS Upon M. avium Infection in the Presence ofAntibiotics

[0147] In order to assess whether ISS would work effectively with otheranti-mycobacterial agents, the effect of ISS and the antibioticclarithromycin (ZITHROMAX®, Pfizer Labs, New York, N.Y.) werecoadministered to M. avium-infected hMDM and intracellular growth of M.avium was evaluated. M. avium-infected hMDM were prepared as describedabove (6×10⁴ hMDM/well), and then incubated with ISS (10 μg/ml) in thepresence or absence of 1 μg/ml, 4 μg/ml, or 20 μg/ml clarithromycin, anantibiotic used to treat M. avium infection. CFU was determined on days0 and 7 after infection. The experiment was done in triplicate andresults expressed as mean±SD of CFU per well.

[0148] On day 7, ISS alone inhibited intracellular growth of M. avium by38±7% (p=0.05) (FIG. 3). ISS further enhanced the anti-mycobacterialeffect of 1 μg/ml and 4 μg/ml clarithromycin by 89±5% (p<0.001) and63±12% (p=0.001), respectively, compared to antibiotic alone. These datashow that ISS and clarithromycin had a synergistic effect in inhibitionof M. avium replication. In another experiment, hMDM (5×10⁴ hMDM/well)were treated with ISS or M-ODN (3, 10, and 30 μg/ml) for 3 days and theninfected them with M. avium. There was maximal inhibition of M. aviumgrowth at 3 μg/ml of ISS (FIG. 4A) with no further increase ininhibition at the higher concentrations (data not shown). At 7 dayspost-infection, treatment with ISS was found to have inhibitedintracellular growth of M. avium by 91% (FIG. 4A, p<0.001). No changesin cell viability in the various groups was observed. To study thetherapeutic effects of ISS on established M. avium infection, infectedhMDM were treated with ISS (10 μg/ml) for 7 days. Treatment with ISSsignificantly decreased the intracellular growth of M. avium in hMDM by53% (p<0.05) (FIG. 4B). When infected cells were treated with ISStogether with CLA (0.5 μg/ml), M. avium growth was further inhibited upto 99% (p<0.01), compared to medium alone (FIG. 4B).

Example 4 ISS is a Potent Adjunct to Anti-Mycobacterial Therapy with CLA

[0149] mBMDM (5×10⁴ mBMDM/well) was first infected with M. avium andthen these cells were treated with CLA (0.1 μg/ml) in the presence orabsence of ISS (10 pg/ml) or M-ODN (10 μg/ml) and M. avium growth invitro 7 days after infection was determined. ISS and CLA (0.1 μg/ml),when used individually, reduced bacterial growth in mBMDM by 68% and84%, respectively (p<0.01, FIG. 5A). When ISS was used together withCLA, bacterial counts were further reduced (95%, p<0.01) compared tomedium alone (FIG. 5A).

[0150] C57B1/6 mice were infected intravenously with M. avium (10⁷ CFU)and were treated with a combination of ISS-ODN (50 μg/mouse) and/or CLA(200 mg/kg) one week after infection. Mice were sacrificed 5 weeks afterinfection and CFU in the spleen, liver, and lungs were counted.Treatment with CLA alone decreased bacterial growth in the spleen (4 logreduction, FIG. 5B), liver (4 log reduction, FIG. 5D) and lungs (1.5 logreduction, FIG. 5C). In this therapeutic model, ISS alone did notinhibit the growth of M. avium. However, when ISS was combined with CLA,there was a further reduction of bacterial counts in the spleen (<1 logreduction, p<0.01, FIG. 5B) and especially in the lungs (3 logreduction, p<0.01, FIGS. 5B-5D). These findings show that ISS canenhance the therapeutic efficacy of CLA in the setting of established M.avium infection.

Example 5 In Vivo Effect of ISS on M. avium Infection in Mice

[0151] The ability of ISS to elicit a protective immune response againstmycobacterial infection was tested in an animal model of disseminatedmycobacterial infection. C57B1/6 mice were pretreated intradermally withISS and subsequently infected with 10⁶ or 10⁷ organisms/mouse asdescribed in the Materials and Methods above.

[0152] a) Bacterial Load

[0153] At 2, 4 and 6 weeks after infection, the spleen, liver, and lungswere collected from the M. avium-infected mice (10⁶ organisms/mouse),homogenized and used to determine the number of CFU as described inMaterials and Methods above.

[0154] At week 2, the lungs of M. avium-infected mice pretreated withISS were found to contain a significantly lower number of viablebacteria compared to those of infected mice which received PBS insteadof ISS (p<0.05). In addition, M. avium-infected mice pretreated with ISSwere found to have an almost two logs lower number of bacteria in thespleen (p<0.05) compared to the PBS-pretreated mice (FIG. 6). Theseeffects were maximal at 4 weeks. At week 6, CFU in the spleen of micetreated with ISS equaled that observed in the control mice treated withPBS. Surprisingly, at week 6 the bacterial load in the lungs ofISS-treated mice exceeded that found in the lungs of control mice (FIG.6). This observation suggests that ISS alone does not eradicate themycobacterial infection under the conditions described.

[0155] There was no significant difference in the bacterial loads in theliver of mice pretreated with ISS compared to those recovered from micepretreated with PBS at any of the time points (data not shown). Thus, asingle injection of ISS significantly reduced the mycobacterial growthin the spleen and the lungs, but not in the liver in M. avium-infectedmice. This protective effect was found to persist for up to four weeksafter administration of ISS. These data demonstrate that ISS by itselfcan induce strong protective immunity against mycobacterial infectionseven in the absence of specific DNA sequences which code formycobacterial antigens.

[0156] In another experiment, mice were treated with ISS and infectedintravenously (i.v.) three days later with M. avium (10⁷organisms/mouse). At 2, 4, and 6 weeks after infection, the number ofCFU in the spleen, lungs, and liver were determined. Four weeks afterinfection, CFU in the spleen and lungs were similar in the mice treatedwith M-ODN and control (PBS) mice, but were significantly higher than inthe ISS-treated mice (by 2 logs and 1 log in spleen and lungs,respectively).

[0157] At week 2, the lungs of M. avium-infected mice treated with ISSprior to infection contained a significantly lower number of viablebacteria compared to control PBS-treated mice (p<0.05) (FIG. 7B). Inaddition, mice treated with ISS prior to M. avium infection had nearlytwo logs less bacteria in the spleen at 4 weeks (p<0.05) compared to thePBS-treated mice (FIG. 7A). By 6 weeks, however, splenic CFU counts weresimilar in control and ISS groups. There was no significant differencein the mycobacterial loads in the liver of mice treated with ISS priorto infection compared to PBS-treated mice at any of these time points(FIG. 7C). Thus, a single injection of ISS significantly reduced themycobacterial growth in the spleen and lungs, but not in the liver of M.avium-infected mice. This protective effect was transient and was mostapparent at 2 and 4 weeks after a single administration of ISS.

[0158] b) Serum IFN-γ and IL-12 Levels.

[0159] To determine whether production of IFN-γ and IL-12 could beresponsible for the protective effect exerted by ISS, serum wascollected from the ISS-treated or PBS-treated M. avium-infected mice(10⁶ organisms/mouse) at 2, 4, and 6 weeks after infection. Serum IFN-γlevels of mice infected with M. avium was found to be significantlyhigher than that of uninfected mice throughout the experiment (p<0.05)(FIG. 8). However, there were no significant differences in the serumIFN-γ levels of ISS-pretreated mice compared to PBS-pretreated mice.Serum IL-12 levels were found to be less than that detectable by theELISA (<5 pg/ml).

[0160] In other experiments not described here, the serum level of IFN-γand IL-12 after administration of ISS peaks at day 1, after which levelsbegin to decline and attain basal levels within 3 weeks post injection(data not shown) (see, e.g., Kobayashi et al. (1999) Cell. Immunol.198:69-25).

[0161] These data indicate that there is no significant difference inserum IFN-γ levels between ISS-treated and PBS-treated infected mice.This could be due to the fact that induction of IFN-γ production is anearly event during the course of M. avium infection. Our earliest bloodsamples for IFN-γ assay were collected 2 weeks after infection, by whichtime the IFN-γ levels may have returned to the basal level.Alternatively, M. avium may induce IFN-7 production as efficiently asISS so no differences would be observed. Serum IL-12 (p70) was found tobe undetectable at all the time points of the experiment (2, 4 and 6weeks). In studies by other investigators, total IL-12 (p4⁰ and p70) wasmeasured, whereas in our study, only biological-active IL-12 p70 wasmeasured, which may explain the disparity in observations.

[0162] c) Histology.

[0163] Tissue sections from the spleen of M. avium-infected mice treatedwith ISS, PBS or uninfected mice were fixed and stained withhematoxylin-eosin as described in the Materials and Methods above. Atweek 4, the white pulp in the spleen from M. avium-infected mice treatedwith PBS was disrupted with the formation of several granulomas, whilethe white pulp in the spleen of the uninfected mice was intact. Sectionsof the spleen from the ISS-treated mice also revealed the presence ofgranulomas, although they were notably smaller in size and surrounded bymononuclear cells in contrast to the spleens from infected mice treatedwith PBS.

[0164] The red pulp in spleens of ISS-treated mice appeared to containincreased hematopoietic cells. However, at week 6, the granulomas in thespleens from ISS-treated, M. avium-infected mice were not significantlydifferent from those of PBS-treated, M. avium-infected mice correlatingwell with the CFU data and the loss of effect of ISS observed at thistime point (presented above).

[0165] Overall, ISS appears to cause a delay in the formation ofgranulomas, which may be associated with the presence of increasednumbers of mononuclear and hematopoietic cells in the spleen. The liversof the M. avium-infected mice showed a significant number of granulomasat weeks 4 and 6, while the livers from uninfected mice did not show anygranulomas, as expected. However, there were no significanthistopathological differences in the livers recovered from theISS-treated, M. avium-infected mice compared to those recovered fromPBS-treated, M. avium-infected mice (data not shown).

[0166] d) Summary

[0167] These data show that a significant inhibitory effect by ISSagainst M. avium growth was seen in the spleen and the lungs, but notsignificantly in the liver. Histopathology studies showed that thespleen of ISS treated, M. avium-infected mice contained significantlyincreased numbers of mononuclear cells compared to the spleen of thePBS-treated, M. avium-infected control mice. No significanthistopathological differences were observed between the livers recoveredfrom ISS-treated and PBS-treated mice.

Example 6 In Vivo Effect of ISS on the Growth of M. avium in Mouse BMDM

[0168] The following studies were performed to address whetherISS-induced activation of macrophages in vitro inhibits intracellulargrowth of M. avium.

[0169] a) Treatment Prior to Infection.

[0170] To examine whether treatment with ISS can stimulate macrophagesto inhibit the growth of M. avium, mBMDM (5×10⁴ mBMDM/well) was firsttreated with ISS or M-ODN for 72 hrs and then infected with M. avium.Then, cellular CFU was counted on days 1, 3, and 7 post-infection (FIG.9A). By day 7, treatment with ISS inhibited intracellular growth of M.avium in mBMDM by 80% (p<0.001). Since viability of macrophages canaffect M. avium growth, the viability of macrophages was assessed bytrypan-blue exclusion. At day 7 after infection, mBMDM treated with ISS,M-ODN or medium alone were all >90% viable.

[0171] ISS activate macrophages and induce the expression of surfaceadhesion molecules such as ICAM-1 (Martin-Orozco, et al. (1999) Int.Immun. 11: 1111-1118). These molecules may affect the attachment of M.avium or its invasion into mouse bone marrow-derived macrophages(mBMDM). In order to determine whether the ability of ISS to inhibit M.avium growth is due to alterations in susceptibility to M. aviuminvasion, the ability of ISS to influence the number of bacteria thatattached to and invaded macrophages after incubation with M. avium wasexamined. CFU recovered immediately from cells treated with ISS beforeinfection were not significantly different than CFU recovered from cellstreated with mutated (M)-ODN or with medium alone.

[0172] b) Treatment After Infection.

[0173] To study the therapeutic anti-mycobacterial effect of ISS,infected mBMDM were treated with ISS for 7 days after infection,starting two hours after time of infection. Treatment with ISSsignificantly decreased the intracellular growth of M. avium in mBMDM by68% (p<0.05), compared to CFU in infected cells treated with M-ODN ormedium alone (FIG. 9B).

Example 7 ISS Protection In Vivo is not Significantly Mediated ThroughAugmentation of the T Cell Response.

[0174] The observations that ISS protects isolated macrophages in vitro(FIG. 9) and that the protective effect observed in ISS-treated mice istransient (FIGS. 6 and 7) suggest a T-cell independent mechanism ofprotection via innate immunity. To further investigate the potentialrole of adaptive immunity in this model of ISS-mediated protectionagainst M. avium, mice were treated with ISS or M-ODN (50 μg/mouse),infected with 10⁷ organisms/mouse, and then their T-cell response wasevaluated. The mice were sacrificed at three weeks post-infection andthe splenocytes were re-stimulated with anti-CD3 and anti-CD28antibodies to amplify the response from pre-existing memory andactivated T cells. T cells were then examined for their production ofIFN-γ by two complementary methods. FACS-based intracellular cytokinestaining was used to determine the frequencies of IFN-γ producing CD4⁺(FIG. 10A) and CD8⁺ (FIG. 10B) T cells, and ELISA was used to determinethe total quantity of IFN-γ secreted by CD4⁺ and CD8⁺ T cells combined(FIG. 10C). There was a dramatic increase in the IFN-γ response of theCD4⁺ T cells and in the total IFN-γ produced in the infected vs.uninfected animals, demonstrating that the observed Th1 response isinfection-specific. However, treatment of M. avium-infected animals withISS did not further increase the frequency of infection-specific IFN-γpositive CD4⁺ or CD8⁺ T cells nor did it increase the secretion of totalinfection-specific IFN-γ. Taken together, these data suggest that themechanism of protection by ISS of M. avium-infected animals does notinvolve enhancement of the anti-mycobacterial T-cell response.

Example 8 ISS Inhibition of M. avium Growth in Macrophages isIndependent of iNOS NAPDH Oxidase, IL-12, TNF-α, IFN-α/β, and IFN-γ.

[0175] To further investigate the mechanisms of the anti-mycobacterialeffects of ISS, mice with targeted disruptions of genes known to playroles in M. avium infection were used. Oxygen radicals generated byNADPH oxidase and induction of nitric oxide (NO) by iNOS result inanti-microbial activity against many microorganisms (Miller, et al.(1997) Clin. Microbiol. Rev. 10:1-18; Fang (1997) J. Clin. Invest.99:2818-2825). IL-12, TNF-α, and IFN-γ play important roles in theclearance of M. avium (Kobayashi, et al. (1995) Antimicrob. AgentsChemother. 39:1369-1371; Doherty, et al. (1998) J. Immunol.160:5428-5435; Appelberg, et al. (1995) Clin. Exp. Immunol.101:308-313). Furthermore, macrophages produce IL-12, TNF-α, IFN-α/β,and IFN-γ in response to ISS treatment (Klinman, et al. (1996) Proc.Natl. Acad. Sci. U S A. 93:2879-2883; Roman, et al. (1997) Nat. Med.3:849-854).

[0176] To study the role of these molecules in the anti-mycobacterialeffect of ISS, mBMDM from NADPH oxidase^(−/−), iNOS^(−/−), TNF-α^(−/−),IL-12p40^(−/−), IFN-α/βR^(−/−) and IFN-γR-^(−/−) mice were treated withISS for 3 days and then infected with M. avium. ISS inhibited theintracellular growth of M. avium in MBMDM from these knockout mice by60-85% (p<0.05), similar to wild-type mice (Table 1). These resultsindicate that these gene products (e.g., nitrogen intermediates, oxygenradicals, TNF-α etc.) are not central to the anti-mycobacterial effectinduced by ISS in vitro. TABLE 1 Effect of ISS on M. avium growth inmBMDM from mice with targeted disruption of genes known to play aprotective role against M. avium infection. % of CFU^(a) mBMDM weretreated with Mouse Strain Untreated M-ODN ISS Wild type C57B1/6 100117.2 ± 18.5 18.2 ± 7.3^(b ) Wild type 129S6/SvEV 100 110.0 ± 15.9 17.0± 4.0^(b ) INOS^(−/−) C57B1/6 100 105.0 ± 13.0 37.3 ± 4.6^(b ) NADPHC57B1/6 100 107.0 ± 15.5 11.4 ± 1.5^(b ) oxidase^(−/−) (gp91 phox^(−/−))TNF-α^(−/−) C57B1/6 100 120.0 ± 20.0 24.0 ± 6.0^(b ) IL-12 p40^(−/−)C57B1/6 100 117.6 ± 9.2  26.4 ± 10.1^(b) IFN-αR^(−/−) 129S6/SvEv 100106.9 ± 11.9 10.9 ± 01.7^(b) IFN-γR^(−/−) 129S6/SvEv 100 108.1 ± 10.8 7.6 ± 01.3^(b) # CFU of cells treated with medium alone was consideredas 100%. Mean and standard deviations from three independent experimentsare shown

Example 9 Induction of Indoleamine 2,3-dioxygenase (IDO) Contributes tothe Anti-Mycobacterial Activity of ISS.

[0177] IDO is the rate-limiting enzyme in the catabolism of tryptophan,which thereby limits the availability of this important amino acid toinvading pathogens (Daubener, et al. (1999) Adv. Exp. Med. Biol.467:517-524). To study the potential role of IDO in theanti-mycobacterial effect of ISS, the following were assessed: 1) theinduction of IDO activity as measured by semi-quantitative RT-PCR invivo and in vitro and 2) the abrogation of the anti-mycobacterial effectof ISS by addition of excess L-tryptophan (L-try) or by using acompetitive inhibitor for IDO, 1-methyl-DL-tryptophan (M-try).

[0178] When mice were injected (i.v.) with 50 μg ISS, IDO gene inductionin vivo was observed in the lungs and spleen after 16 hrs, but not inthe liver (FIG. 11A). Injection of M-ODN did not result in anydetectable induction of IDO. For in vitro studies mBMDM were treatedwith ISS for 3 days prior to M. avium infection. Then, cells were lysedat 4, 8, and 24 hrs after infection, total RNA was extracted, andsemi-quantitative RT-PCR was performed. Optimal induction of IDO genetranscription was found to require both treatment with ISS and M. aviuminfection (FIG. l B).

[0179] In order to further investigate the role of IDO in the inhibitionof M. avium growth, mBMDM were cultured with the IDO inhibitor M-try(125 μM) or with excess L-try (final concentration of 66 μg/ml) (Hwu,etal. (2000) J. Immunol. 164:3596-3599; Munn, et al. (1999) J. Exp. Med.189:1363-1372). Addition of L-try or M-try alone at these concentrationsdid not alter the viability of mBMDM or M. avium growth in these cells.However, when the M. avium-infected cells were cultured with L-try orM-try supplemented media, 4-fold reductions in the anti-mycobacterialability of ISS treatment (p<0.05) was observed (FIG. 11C). Takentogether, these data show that IDO plays a major role in the observedanti-mycobacterial properties of ISS.

[0180] IDO inhibits the growth of a variety of intracellular organismssuch as Toxoplasma gondi (Pfefferkorn, et al. (1984) Infect. Immun.44:211-216), Plasmodium berghe in a murine model of malaria (Sanni, etal. (1998) Am. J. Pathol. 152:611-619), Chlamydia psittaci (Carlin, etal. (1989) J. Interferon Res. 9:329-337), and Chlamydia trachomatis(Beatty, et al.(1994) Infect. Immun. 62:3705-3711) by breaking theL-tryptophan required for their growth down to L-kynurenine. IDO hasbeen described to be the most effective anti-parasitic mechanism in mosthuman cells (Daubener, et al. (1999) Med. Micro. Immunol. 187:143-147),indicating the broad applicability of ISS for treatment of infection bya wide variety of intracellular pathogens. The anti-pathogenic effectsof immunomodulatory nucleic acids such as ISS may induce otheranti-pathogen pathways in the host in addition to induction of IDO.

[0181] In summary, this study demonstrates that administration of ISSenhances resistance against M. avium infection through the induction ofIDO. The ISS itself provides protection against M. avium. However thiseffect can be amplified upon co-delivery with an anti-mycobacterialdrug, Clarithromycin. The combined administration of ISS with otherantibiotics or anti-pathogenic agents provides an alternativetherapeutic strategy for intracellular pathogen infections.

[0182] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

That which is claimed is:
 1. A method for treating an intracellularpathogen infection in a subject, the method comprising: administering toa subject an immunomodulatory nucleic acid molecule in an amounteffective to inhibit intracellular replication of the intracellularpathogen; and administering to the subject an anti-pathogenic agent inan amount effective to decrease or inhibit growth of the intracellularpathogen, thereby treating the pathogen.
 2. The method of claim 1,wherein the immunomodulatory nucleic acid molecule is selected from thegroup consisting of an immunostimulatory oligodeoxyribonucleotide(ISS-ODN); an isolated, detoxified bacterial polynucleotide; and anISS-enriched plasmid DNA.
 3. The method of claim 1, wherein theimmunomodulatory nucleic acid molecule comprises a CpG motif selectedfrom the group consisting of: a)5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′; b)5′-Purine-TCG-Pyrimidine-Pyrimidine-3′; c) 5′-[TCG]_(n)-3′, where n isany integer that is 1 or greater; and d)5′-Purine-Purine-CG-Pyrimidine-Pyrimidine-CG-3′. e)5′-Purine-TCG-Pyrimidine-Pyrimidine-CG-3′
 4. The method of claim 1,wherein the immunomodulatory nucleic acid molecule comprises a sequenceselected from the group consisting of: AACGCC, AACGCT, AACGTC, AACGTT,AGCGCC, AGCGCT, AGCGTC, AGCGTT, GACGCC, GACGCT, GACGTC, GACGTT, GGCGCC,GGCGCT, GGCGTC, GGCGTT, ATCGCC, ATCGCT, ATCGTC,ATCGTT,GTCGCC,GTCGCT,GTCGTC,GTCGTT,TCGTCG,TCGTCGTCG,AACGCCCG,AACGCTCG,AACGTCCG,AACGTTCG,AGC.GCCCG,AGCGCTCG,AGCGTCCG,AGCGTTCG,GACGCCCG,GACGCTCG,GACGTCCG,GACGTTCG,GGCGCCCG,GGCGCTCG,GGCGTCCG,GGCGTTCG, ATCGCCCG,ATCGCTCG,ATCGTCCG,ATCGTTCG, GTCGCCCG, GTCGCTCG, GTCGTCCG, and GTCGTTCG.
 5. Themethod of claim 4, wherein the immunomodulatory nucleic acid moleculecomprises the sequence AACGTTCG.
 6. The method of claim 1, wherein theimmunomodulatory nucleic acid molecule is administered in an amounteffective to provide synergistic anti-pathogenic activity with theanti-pathogenic agent.
 7. The method of claim 1, wherein theimmunomodulatory nucleic acid molecule and the anti-pathogenic agent areadministered concurrently.
 8. The method of claim 1, wherein theintracellular pathogen is a bacterium.
 9. The method of claim 8, whereinthe bacterium is a Mycobacterium bacterium.
 10. The method of claim 9,wherein the bacterium is selected from the group consisting ofMycobacterium tuberculosis and Mycobacterium avium.
 11. The method ofclaim 1, wherein said administering enhances indoleamine 2,3-dioxygenaseactivity in the subject.
 12. The method of claim 1, wherein the subjectis immunocompromised.
 13. The method of claim 12, wherein theimmunocompromised subject has a reduced number of CD4+ T cells relativeto an immunocompetent subject.
 14. A method for treating a mycobacterialinfection in a subject, the method comprising: administering to asubject an immunomodulatory nucleic acid molecule in an amount effectiveto inhibit replication of a Mycobacterium bacterium, thereby treatingmycobacterial indection in the subject.
 15. The method of claim 14,wherein the immunomodulatory nucleic acid molecule is selected from thegroup consisting of an immunostimulatory oligodeoxyribonucleotide(ISS-ODN); an isolated, detoxified bacterial polynucleotide; and anISS-enriched plasmid DNA.
 16. The method of claim 14, wherein theimmunomodulatory nucleic acid molecule comprises a CpG motif selectedfrom the group consisting of: a)5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′; b)5′-Purine-TCG-Pyrimidine-Pyrimidine-3′; c) 5′-[TCG]_(n)-3′, where n isany integer that is 1 or greater; and d)5′-Purine-Purine-CG-Pyrimidine-Pyrimidine-CG-3′. e)5′-Purine-TCG-Pyrimidine-Pyrimidine-CG-3′
 17. The method of claim 14,wherein the immunomodulatory nucleic acid molecule comprises a sequenceselected from the group consisting of: AACGCC, AACGCT, AACGTC, AACGTT,AGCGCC, AGCGCT, AGCGTC, AGCGTT, GACGCC, GACGCT, GACGTC, GACGTT, GGCGCC,GGCGCT, GGCGTC, GGCGTT, ATCGCC, ATCGCT, ATCGTC, ATCGTT, GTCGCC, GTCGCT,GTCGTC, GTCGTT, TCGTCG, TCGTCGTCG, AACGCCCG, AACGCTCG, AACGTCCG,AACGTTCG, AGCGCCCG, AGCGCTCG, AGCGTCCG, AGCGTTCG, GACGCCCG, GACGCTCG,GACGTCCG, GACGTTCG, GGCGCCCG, GGCGCTCG, GGCGTCCG, GGCGTTCG, ATCGCCCG,ATCGCTCG, ATCGTCCG, ATCGTTCG, GTCGCCCG, GTCGCTCG, GTCGTCCG, andGTCGTTCG.
 18. The method of claim 17, wherein the immunomodulatorynucleic acid molecule comprises the sequence AACGTTCG.
 19. The method ofclaim 14, wherein said administering results in induction of an immuneresponse effective against infection by a mycobacterial pathogen. 20.The method of claim 14, wherein the immunomodulatory nucleic acidmolecule is administered with an anti-pathogenic agent.
 21. The methodof claim 20, wherein the immunomodulatory nucleic acid molecule isadministered in an amount effective to provide synergisticanti-pathogenic activity with the anti-pathogenic agent.
 22. The methodof claim 20, wherein the immunomodulatory nucleic acid molecule and theanti-pathogenic agent are administered concurrently.
 23. The method ofclaim 14, wherein the bacterium is Mycobacterium tuberculosis.
 24. Themethod of claim 14, wherein the bacterium is Mycobacterim avium.
 25. Themethod of claim 14, wherein the subject is immunocompromised.
 26. Themethod of claim 25, wherein the immunocompromised subject has a reducednumber of CD4+ T cells relative to a immunocompetent subject.
 27. Amethod for inducing in a subject an immune response against aMycobacteriurn bacterium, the method comprising: administering to asubject an amount of an immunomodulatory nucleic acid molecule in anamount effective to elicit an immune response against a Mycobacteriumbacterium; wherein said administering results in induction of an immuneresponse effective to protect the subject against onset of disease or todecrease severity of symptoms of disease caused by infection by theMycobacterium bacterium.
 28. The method of claim 27, wherein theimmunomodulatory nucleic acid molecule comprises a CpG motif selectedfrom the group consisting of: a)5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′; b)5′-Purine-TCG-Pyrimidine-Pyrimidine-3′; c) 5′-[TCG]α-3′, where n is anyinteger that is 1 or greater; and d)5′-Purine-Purine-CG-Pyrimidine-Pyrimidine-CG-3′. e)5′-Purine-TCG-Pyrimidine-Pyrimidine-CG-3′
 29. The method of claim 27,wherein the immunomodulatory nucleic acid molecule comprises a sequenceselected from the group consisting of: AACGCC, AACGCT, AACGTC, AACGTT,AGCGCC, AGCGCT, AGCGTC, AGCGTT, GACGCC, GACGCT, GACGTC, GACGTT, GGCGCC,GGCGCT, GGCGTC, GGCGTT, ATCGCC, ATCGCT, ATCGTC, ATCGTT, GTCGCC, GTCGCT,GTCGTC, GTCGTT, TCGTCG, TCGTCGTCG, AACGCCCG, AACGCTCG, AACGTCCG,AACGTTCG, AGCGCCCG, AGCGCTCG, AGCGTCCG, AGCGTTCG, GACGCCCG, GACGCTCG,GACGTCCG, GACGTTCG, GGCGCCCG, GGCGCTCG, GGCGTCCG, GGCGTTCG, ATCGCCCG,ATCGCTCG, ATCGTCCG, ATCGTTCG, GTCGCCCG, GTCGCTCG, GTCGTCCG, andGTCGTTCG.
 30. The method of claim 29, wherein the immunomodulatorynucleic acid molecule comprises the sequence AACGTTCG.
 31. The method ofclaim 27, wherein the bacterium is Mycobacterium tuberculosis.
 32. Themethod of claim 27, wherein the bacterium is Mycobacterium avium. 33.The method of claim 27, wherein the subject is immunocompromised. 34.The method of claim 33, wherein the immunocompromised subject has areduced number of CD4+ T cells relative to an immunocompetent subject.35. A method treating a mycobacterial infection, the method comprising:administering to a subject an amount of an immunomodulatory nucleic acidmolecule in an amount effective to inhibit intracellular replication ofa Mycobacterium bacterium in the subject; and administering to thesubject an antimicrobial agent in an amount effective to decrease orinhibit growth of the Mycobacterium bacterium; wherein saidadministering is effective to decrease severity of symptoms of diseasecaused by the Mycobacterium bacterium.
 36. The method of claim 35,wherein the immunomodulatory nucleic acid molecule is administered in anamount effective to provide a synergistic, antimicrobial effect with theantimicrobial agent.
 37. The method of claim 35, wherein theimmunomodulatory nucleic acid comprises a sequence selected from thegroup consisting of: AACGCC, AACGCT, AACGTC, AACGTT, AGCGCC, AGCGCT,AGCGTC, AGCGTT, GACGCC, GACGCT, GACGTC, GACGTT, GGCGCC, GGCGCT, GGCGTC,GGCGTT, ATCGCC, ATCGCT, ATCGTC, ATCGTT, GTCGCC, GTCGCT, GTCGTC, GTCGTT,TCGTCG, TCGTCGTCG, AACGCCCG, AACGCTCG, AACGTCCG, AACGTTCG, AGCGCCCG,AGCGCTCG, AGCGTCCG, AGCGTTCG, GACGCCCG, GACGCTCG, GACGTCCG, GACGTTCG,GGCGCCCG, GGCGCTCG, GGCGTCCG, GGCGTTCG, ATCGCCCG, ATCGCTCG, ATCGTCCG,ATCGTTCG, GTCGCCCG, GTCGCTCG, GTCGTCCG, and GTCGTTCG.
 38. The method ofclaim 37, wherein the immunomodulatory nucleic acid comprises thesequence AACGTTCG.
 39. The method of claim 35, wherein saidadministering of the immunomodulatory nucleic acid enhances indoleamine2,3-dioxygenase activity in the subject.
 40. A method for treating anintracellular pathogen infection in a subject, the method comprising:administering to a subject an immunomodulatory nucleic acid molecule inan amount effective to enhance indoleamine 2,3-dioxygenase activity inthe subject, thereby inhibiting intracellular replication by anintracellular pathogen and treating infection in the subject.
 41. Themethod of claim 40, wherein the immunomodulatory nucleic acid moleculeis selected from the group consisting of an immunostimulatoryoligodeoxyribonucleotide (ISS-ODN); an isolated, detoxified bacterialpolynucleotide; and an ISS-enriched plasmid DNA.
 42. The method of claim40, wherein the immunomodulatory nucleic acid molecule comprises a CpGmotif selected from the group consisting of: a)5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′; b)5′-Purine-TCG-Pyrimidine-Pyrimidine-3′; c) 5′-[TCG]_(n),-3′, where n isany integer that is 1 or greater; and d)5′-Purine-Purine-CG-Pyrimidine-Pyrimidine-CG-3′. e)5′-Purine-TCG-Pyrimidine-Pyrimidine-CG-3′
 43. The method of claim 40,wherein the immunomodulatory nucleic acid molecule comprises a sequenceselected from the group consisting of: AACGCC, AACGCT, AACGTC, AACGTT,AGCGCC, AGCGCT, AGCGTC, AGCGTT, GACGCC, GACGCT, GACGTC, GACGTT, GGCGCC,GGCGCT, GGCGTC, GGCGTT, ATCGCC, ATCGCT, ATCGTC, ATCGTT, GTCGCC, GTCGCT,GTCGTC, GTCGTT, TCGTCG, TCGTCGTCG, AACGCCCG, AACGCTCG, AACGTCCG,AACGTTCG, AGCGCCCG, AGCGCTCG, AGCGTCCG, AGCGTTCG, GACGCCCG, GACGCTCG,GACGTCCG, GACGTTCG, GGCGCCCG, GGCGCTCG, GGCGTCCG, GGCGTTCG, ATCGCCCG,ATCGCTCG, ATCGTCCG, ATCGTTCG, GTCGCCCG, GTCGCTCG, GTCGTCCG, andGTCGTTCG.
 44. The method of claim 43, wherein the immunomodulatorynucleic acid molecule comprises the sequence AACGTTCG.
 45. The method ofclaim 40, wherein the immunomodulatory nucleic acid molecule isadministered with an anti-pathogenic agent.
 46. The method of claim 45,wherein the immunomodulatory nucleic acid molecule is administered in anamount effective to provide synergistic anti-pathogenic activity withthe anti-pathogenic agent.
 47. The method of claim 45, wherein theimmunomodulatory nucleic acid molecule and the anti-pathogenic agent areadministered concurrently.
 48. The method of claim 40, wherein theintracellular pathogen is a Mycobacterium bacterium.
 49. The method ofclaim 48, wherein the bacterium is selected from the group consisting ofMycobacterium tuberculosis and Mycobacterium avium.
 50. The method ofclaim 40, wherein the subject is immunocompromised.
 51. The method ofclaim 50, wherein the immunocompromised subject has a reduced number ofCD4+ T cells relative to an immunocompetent subject.